Materials and methods for performing histochemical assays for human pro-epiregulin and amphiregulin

ABSTRACT

The invention provides anti-human pro-epiregulin and anti-human amphiregulin antibodies and methods of using the same. Anti-EREG antibodies raised against amino acids 148-169 and 156-169 of the human EREG protein, and anti-AREG antibodies raised against amino acids 238-252 of the human AREG protein are disclosed. Methods of using these antibodies to detect EREG and AREG and kits and other products for performing such methods are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 15/851,502,filed Dec. 21, 2017, which is a continuation of PCT/EP2016/064883, filedJun. 27, 2016, which claims the benefit of U.S. Provisional PatentApplication No. 62/186,251, filed Jun. 29, 2015, the content of each ofwhich is incorporated by reference herein in its entirety.

SEQUENCE LISTING INCORPORATION BY REFERENCE

This application hereby incorporates-by-reference a sequence listingsubmitted herewith in a computer-readable format, having a file name ofP32945US2_SEQLIST_ST25, created on Oct. 19, 2020, which is 20,944 bytesin size.

BACKGROUND OF THE INVENTION A. Technical Field

The present invention relates to antibodies for detecting epiregulin andamphiregulin in human samples and methods of using the same.

B. Description of Related Art

About 20% of patients with colon cancer present with metastaticcolorectal cancer (mCRC) but regardless of the treatment they receivemore than half (50-60%) of these patients will eventually developincurable advanced disease, which has a 5 year survival rate ofapproximately 12.5%. Two signaling pathways in mCRC have been the focusof therapeutic drug development: the vascular endothelial growth factorreceptor (VEGFR) and the epidermal growth factor receptor (EGFR)pathways. Currently, the majority of the patients with mCRC receivecytotoxic chemotherapy combined with either EGFR or VEGF-targetedtherapies. EGFR is overexpressed in about 70% of CRC cases where it isassociated with poor outcome. Targeted inhibition of EGFR withmonoclonal antibodies, cetuximab or panitumumab, was approved by FDA in2004 and 2006 to treat patients with mCRC. These antibodies target theextracellular domain of EGFR and compete with endogenous ligands toprevent activation of the receptor. By inhibiting EGFR signaling pathwaythese biological agents inhibit cell proliferation, differentiation,migration and metastasis. Both drugs have very similar efficacy with a10-15% response rate.

Several molecular markers have been investigated to better predictresponse to anti-EGFR therapy. See Perkins et al., Pharmacogenetics,Vol. 15, Issue 7, pp. 1043-52 (2014). Clinical studies have providedevidence that EGFR inhibitors are the most effective in patients lackingRAS pathway mutations and maybe detrimental to those who have mutanttype tumor. Point mutations in members of the RAS signaling pathwayssuch as KRAS, NRAS, or BRAF lead to continuous activation downstreamRAS-MAPK signaling, regardless of whether the EGFR is pharmacologicallyinactivated. In addition to RAS and BRAF mutations, other alternativemechanisms such as cMET or EGFR amplification play a role in resistanceto Cetuximab or Panitumumab. PI3K-AKT-PTEN pathway can also be triggeredby EGFR activation therefore mutation in PI3K or PTEN loss (often occurwith KRAS or BRAF mutations) is also associated with a lack of response.RAS, BRAF, and PI3K mutations account for more than 60% of patients withmCRC that show de novo resistance to EGFR-targeted monoclonalantibodies. Of the 40% of patients with KRAS, NRAS, BRAF and PI3K wildtype tumors (quadruple wild type patients), approximately half of thesepatients (only 15%) have a major benefit from anti-EGFR therapy and morethan 20% are non-responders. Since RAS, RAF, PI3K status is notsufficient to evaluate anti-EGFR response; there is an unmet medicalneed to improve patients'selection for anti-EGFR therapy.

Several potential candidates are under investigations that are involvedeither in EGFR signaling pathway or in other pathways as MET or HERreceptors. Elevated gene expression of epiregulin (EREG) and/oramphiregulin (AREG), ligands for EGFR has been consistently proposed forprediction of anti-EGFR therapy. In these tumors, anti-EGFR antibodiesare competing with ligand-dependent activation of EGFR, leading to downregulation of the receptor from the cell surface, thus suppressingproliferative signaling. One recently published study showed thatpatients whose tumors had low EREG mRNA levels had no benefit fromanti-EGFR therapy; the cetuximab therapy was not associated with animprovement in overall survival (OS). While in the biomarker positivegroup (KRAS wt/EREG high mRNA) the increased EREG mRNA expression wasstrongly associated with increased therapeutic benefit from cetuximab.In terms of absolute median OS gain, the addition of anti-EGFR therapyincreased survival from 5.1 to 9.8 months compared to the bestsupportive care alone. This result suggests that EGFR ligands expressionmight become a clinically useful biomarker to screen patients with mCRCfor EGFR inhibitor therapy.

It therefore would be useful to have new antibodies available fordetection of EGFR ligands, such as EREG and AREG, in tissue samples.

SUMMARY OF THE INVENTION

The present disclosure relates to anti-pro-epiregulin antibodies,anti-amphiregulin antibodies, and methods of using the same.

In one aspect, an antibody, antigen-binding fragment thereof, or arecombinant protein thereof is disclosed, wherein the antibody iscapable of specifically binding to human pro-epiregulin, such as a humanpro-epiregulin molecule according to SEQ ID NO: 1.

In one aspect, an antibody, antigen-binding fragment thereof, or arecombinant protein thereof is disclosed, wherein the antibody iscapable of specifically binding to amino acids 148-169 of SEQ ID NO: 1.

In one aspect, an antibody, antigen-binding fragment thereof, or arecombinant protein thereof is disclosed, wherein the antibody iscapable of specifically binding to human pro-epiregulin, wherein theantibody binds to an epitope comprising amino acid residues 148-169 ofhuman pro-epiregulin polypeptide according to SEQ ID NO: 1. In someembodiments, the antibody comprises the following hypervariable regions(HVRs): (a) an HVR-H1 comprising the amino acid sequence of RYGMS (SEQID NO: 2); (b) an HVR-H2 comprising the amino acid sequence ofSINRTAYTYYATWAKG (SEQ ID NO: 3); and (c) an HVR-H3 comprising the aminoacid sequence of GLTYGGSDYDYDDAL (SEQ ID NO: 4). In some embodiments,the antibody further comprises the following heavy chain variable domainframework regions (FRs): (a) FR-H1 comprising the amino acid sequence ofQSVEESGGRLVTPGTPLTLTCTVSGFSLS (SEQ ID NO: 5); (b) FR-H2 comprising theamino acid sequence of WVRQAPGKGLEYIG (SEQ ID NO: 6); (c) FR-H3comprising the amino acid sequence of RFTISRTSTTVDLRMTSLTTEDTATYFCAR(SEQ ID NO: 7); and (d) FR-H4 comprising the amino acid sequence ofWGPGTLVTVSS (SEQ ID NO: 8). In some embodiments, the antibody furthercomprises the following HVRs: (a) an HVR-L1 comprising the amino acidsequence of QASQSVYKNKNLA (SEQ ID NO: 9); (b) an HVR-L2 comprising theamino acid sequence of RASTLAS (SEQ ID NO: 10); and (c) an HVR-L3comprising the amino acid sequence of QGEFSCSTFDCIL (SEQ ID NO: 11). Insome embodiments, the antibody further comprises the following lightchain variable domain FRs: (a) FR-L1 comprising the amino acid sequenceof QVLTQTPSSVSAAVGGTVTINC (SEQ ID NO: 12); (b) FR-L2 comprising theamino acid sequence of WYQQKPGQPPKLLIY (SEQ ID NO: 13); (c) FR-L3comprising the amino acid sequence of GVSSRFKGSGSGTQFTLTISGVQCADAATYYC(SEQ ID NO: 14); and (d) FR-L4 comprising the amino acid sequence ofFGGGTEMVVK (SEQ ID NO: 15). In some embodiments, the antibody comprises(a) a VH sequence having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO: 16; (b) a VL sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO: 17; or (c) aVH sequence as in (a) and a VL sequence as in (b). In some embodiments,the antibody comprises a VH sequence of SEQ ID NO: 16. In someembodiments, the antibody comprises a VL sequence of SEQ ID NO: 17.

In other embodiments, the antibody comprises the following HVRs: (a) anHVR-L1 comprising the amino acid sequence of QASQSVYKNKNLA (SEQ ID NO:9); (b) an HVR-L2 comprising the amino acid sequence of RASTLAS (SEQ IDNO: 10); and (c) an HVR-L3 comprising the amino acid sequence ofQGEFSCSTFDCIL (SEQ ID NO: 11). In some embodiments, the antibody furthercomprises the following light chain variable domain FRs: (a) FR-L1comprising the amino acid sequence of QVLTQTPSSVSAAVGGTVTINC (SEQ ID NO:12); (b) FR-L2 comprising the amino acid sequence of WYQQKPGQPPKLLIY(SEQ ID NO: 13); (c) FR-L3 comprising the amino acid sequence ofGVSSRFKGSGSGTQFTLTISGVQCADAATYYC (SEQ ID NO: 14); and (d) FR-L4comprising the amino acid sequence of FGGGTEMVVK (SEQ ID NO: 15).

In another aspect, the invention features an isolated antibody thatspecifically binds human pro-epiregulin, wherein the antibody comprisesthe following HVRs: (a) an HVR-H1 comprising the amino acid sequence ofRYGMS (SEQ ID NO: 2); (b) an HVR-H2 comprising the amino acid sequenceof SINRTAYTYYATWAKG (SEQ ID NO: 3); (c) an HVR-H3 comprising the aminoacid sequence of GLTYGGSDYDYDDAL (SEQ ID NO: 4); (d) an HVR-L1comprising the amino acid sequence of QASQSVYKNKNLA (SEQ ID NO: 9); (e)an HVR-L2 comprising the amino acid sequence of RASTLAS (SEQ ID NO: 10);and (f) an HVR-L3 comprising the amino acid sequence of QGEFSCSTFDCIL(SEQ ID NO: 11). In some embodiments, the antibody further comprises thefollowing heavy chain variable domain and light chain variable domainFRs: (a) FR-H1 comprising the amino acid sequence ofQSVEESGGRLVTPGTPLTLTCTVSGFSLS (SEQ ID NO: 5); (b) FR-H2 comprising theamino acid sequence of WVRQAPGKGLEYIG (SEQ ID NO: 6); (c) FR-H3comprising the amino acid sequence of RFTISRTSTTVDLRMTSLTTEDTATYFCAR(SEQ ID NO: 7); (d) FR-H4 comprising the amino acid sequence ofWGPGTLVTVSS (SEQ ID NO: 8); (e) FR-L1 comprising the amino acid sequenceof QVLTQTPSSVSAAVGGTVTINC (SEQ ID NO: 12); (f) FR-L2 comprising theamino acid sequence of WYQQKPGQPPKLLIY (SEQ ID NO: 13); (g) FR-L3comprising the amino acid sequence of GVSSRFKGSGSGTQFTLTISGVQCADAATYYC(SEQ ID NO: 14); and (h) FR-L4 comprising the amino acid sequence ofFGGGTEMVVK (SEQ ID NO: 15). In some embodiments, the antibody comprisesa VH sequence of SEQ ID NO: 16 and a VL sequence of SEQ ID NO: 17.

In one aspect, an antibody, antigen-binding fragment thereof, or arecombinant protein thereof is disclosed, wherein the antibody iscapable of specifically binding to amino acids 156-169 of SEQ ID NO: 1.

In one aspect, an antibody, antigen-binding fragment thereof, or arecombinant protein thereof is disclosed, wherein the antibody iscapable of specifically binding to human pro-epiregulin, wherein theantibody binds to an epitope comprising amino acid residues 156-169 ofhuman pro-epiregulin polypeptide according to SEQ ID NO: 1. In someembodiments, the antibody comprises the following hypervariable regions(HVRs): (a) an HVR-H1 comprising the amino acid sequence of TFAMA (SEQID NO: 18); (b) an HVR-H2 comprising the amino acid sequence ofFISLSDATYYATWAKG (SEQ ID NO: 19); and (c) an HVR-H3 comprising the aminoacid sequence of VVGDSSGYPNTFHP (SEQ ID NO: 20). In some embodiments,the antibody further comprises the following heavy chain variable domainframework regions (FRs): (a) FR-H1 comprising the amino acid sequence ofKSVEESGGRLVTPGTPLTLTCTVSGIDLS (SEQ ID NO: 21); (b) FR-H2 comprising theamino acid sequence of WVRQAPGKGLEYIG (SEQ ID NO: 22); (c) FR-H3comprising the amino acid sequence of RFTISKSSSTTVDLKIITPTAEDTATYFCAR(SEQ ID NO: 23); and (d) FR-H4 comprising the amino acid sequence ofWGPGTLVTVSS (SEQ ID NO: 24). In some embodiments, the antibody furthercomprises the following HVRs: (a) an HVR-L 1 comprising the amino acidsequence of QASQSIHNSDFLA (SEQ ID NO: 25) or QASQNIHNSDFLA (SEQ ID NO:26); (b) an HVR-L2 comprising the amino acid sequence of RASKLPS (SEQ IDNO: 27); and (c) an HVR-L3 comprising the amino acid sequence ofQGTYYSGGWYFT (SEQ ID NO: 28). In some embodiments, the antibody furthercomprises the following light chain variable domain FRs: (a) FR-L1comprising the amino acid sequence of QVLTQTPSPVSAAVGGTVTINC (SEQ ID NO:29); (b) FR-L2 comprising the amino acid sequence of WYQQKPGQPPKLLIY(SEQ ID NO: 30); (c) FR-L3 comprising the amino acid sequence ofGVPSRFKGSGSGTQFTLTISDLECDDAATYYC (SEQ ID NO: 31); and (d) FR-L4comprising the amino acid sequence of FGGGTEVVVK (SEQ ID NO: 32). Insome embodiments, the antibody comprises (a) a VH sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO: 33;(b) a VL sequence having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO: 34 or SEQ ID NO: 35; or (c) a VH sequence asin (a) and a VL sequence as in (b). In some embodiments, the antibodycomprises a VH sequence of SEQ ID NO: 33. In some embodiments, theantibody comprises a VL sequence of SEQ ID NO: 34 or SEQ ID NO: 35. Insome embodiments, the antibody comprises a VH sequence of SEQ ID NO: 33and a VL sequence of SEQ ID NO: 34. In some embodiments, the antibodycomprises a VH sequence of SEQ ID NO: 33 and a VL sequence of SEQ ID NO:35.

In other embodiments, the antibody comprises the following HVRs: (a) anHVR-L1 comprising the amino acid sequence of QASQSIHNSDFLA (SEQ ID NO:25) OR QASQNIHNSDFLA (SEQ ID NO: 26); (b) an HVR-L2 comprising the aminoacid sequence of RASKLPS (SEQ ID NO: 27); and (c) an HVR-L3 comprisingthe amino acid sequence of QGTYYSGGWYFT (SEQ ID NO: 28). In someembodiments, the antibody further comprises the following light chainvariable domain FRs: (a) FR-L1 comprising the amino acid sequence ofQVLTQTPSPVSAAVGGTVTINC (SEQ ID NO: 29); (b) FR-L2 comprising the aminoacid sequence of WYQQKPGQPPKLLIY (SEQ ID NO: 30); (c) FR-L3 comprisingthe amino acid sequence of GVPSRFKGSGSGTQFTLTISDLECDDAATYYC (SEQ ID NO:31); and (d) FR-L4 comprising the amino acid sequence of FGGGTEVVVK (SEQID NO: 32).

In another aspect, the invention features an isolated antibody thatspecifically binds human pro-epiregulin, wherein the antibody comprisesthe following HVRs: (a) an HVR-H1 comprising the amino acid sequence ofTFAMA (SEQ ID NO: 18); (b) an HVR-H2 comprising the amino acid sequenceof FISLSDATYYATWAKG (SEQ ID NO: 19); (c) an HVR-H3 comprising the aminoacid sequence of VVGDSSGYPNTFHP (SEQ ID NO: 20); (d) an HVR-L1comprising the amino acid sequence of QASQSIHNSDFLA (SEQ ID NO: 25) ORQASQNIHNSDFLA (SEQ ID NO: 26); (e) an HVR-L2 comprising the amino acidsequence of RASKLPS (SEQ ID NO: 27); and (f) an HVR-L3 comprising theamino acid sequence of QGTYYSGGWYFT (SEQ ID NO: 28). In someembodiments, the antibody further comprises the following heavy chainvariable domain and light chain variable domain FRs: (a) FR-H1comprising the amino acid sequence of KSVEESGGRLVTPGTPLTLTCTVSGIDLS (SEQID NO: 21); (b) FR-H2 comprising the amino acid sequence ofWVRQAPGKGLEYIG (SEQ ID NO: 22); (c) FR-H3 comprising the amino acidsequence of RFTISKSSSTTVDLKIITPTAEDTATYFCAR (SEQ ID NO: 23); (d) FR-H4comprising the amino acid sequence of WGPGTLVTVSS (SEQ ID NO: 24); (e)FR-L1 comprising the amino acid sequence of QVLTQTPSPVSAAVGGTVTINC (SEQID NO: 29); (f) FR-L2 comprising the amino acid sequence ofWYQQKPGQPPKLLIY (SEQ ID NO: 30); (g) FR-L3 comprising the amino acidsequence of GVPSRFKGSGSGTQFTLTISDLECDDAATYYC (SEQ ID NO: 31); and (h)FR-L4 comprising the amino acid sequence of FGGGTEVVVK (SEQ ID NO: 32).In some embodiments, the antibody comprises a VH sequence of SEQ ID NO:33 and a VL sequence of SEQ ID NO: 34 or SEQ ID NO: 35.

In another aspect, the invention features an isolated antibody thatcompetes for binding to human pro-epiregulin with any one of thepreceding anti-pro-epiregulin antibodies.

In one aspect, an antibody, antigen-binding fragment thereof, or arecombinant protein thereof is disclosed, wherein the antibody iscapable of specifically binding to amphiregulin.

In one aspect, an antibody, antigen-binding fragment thereof, or arecombinant protein thereof is disclosed, wherein the antibody iscapable of specifically binding to amino acids 238-252 of SEQ ID NO: 36.

In one aspect, an antibody, antigen-binding fragment thereof, or arecombinant protein thereof is disclosed, wherein the antibody iscapable of specifically binding to amphiregulin, wherein the antibodybinds to an epitope comprising amino acid residues 238-252 of a humanamphiregulin polypeptide (such as SEQ ID NO: 36). In some embodiments,the antibody comprises the following hypervariable regions (HVRs): (a)an HVR-H1 comprising the amino acid sequence of SYAIS (SEQ ID NO: 37);(b) an HVR-H2 comprising the amino acid sequence of FIVGSSGSAYYASWAKS(SEQ ID NO: 38); and (c) an HVR-H3 comprising the amino acid sequence ofGLYSGGNY (SEQ ID NO: 39). In some embodiments, the antibody furthercomprises the following heavy chain variable domain framework regions(FRs): (a) FR-H1 comprising the amino acid sequence ofQSLEESRGGLIKPGGTLTLTCTVSGFSLS (SEQ ID NO: 40); (b) FR-H2 comprising theamino acid sequence of WVRQAPGNGLEWIG (SEQ ID NO: 41); (c) FR-H3comprising the amino acid sequence of RSTITRDTNLNTVTLKMTSLTAADTATYFCAK(SEQ ID NO: 42); and (d) FR-H4 comprising the amino acid sequence ofWGPGTLVTVSS (SEQ ID NO: 43). In some embodiments, the antibody furthercomprises the following HVRs: (a) an HVR-L1 comprising the amino acidsequence of QSSQSVDENNYLS (SEQ ID NO: 44); (b) an HVR-L2 comprising theamino acid sequence of RASTLES (SEQ ID NO: 45); and (c) an HVR-L3comprising the amino acid sequence of LGGYSGYSDDG (SEQ ID NO: 46). Insome embodiments, the antibody further comprises the following lightchain variable domain FRs: (a) FR-L1 comprising the amino acid sequenceof AVLTQTPSPVSAAVGGTVSISC (SEQ ID NO: 47); (b) FR-L2 comprising theamino acid sequence of WFQQKPGQPPKLLIY (SEQ ID NO: 48); (c) FR-L3comprising the amino acid sequence of GVPSRFSGSGSGTQFTLTVSGVQCDDAATYYC(SEQ ID NO: 49); and (d) FR-L4 comprising the amino acid sequence ofFGGGTEVVVK (SEQ ID NO: 50). In some embodiments, the antibody comprises(a) a VH sequence having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO: 51; (b) a VL sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO: 52; or (c) aVH sequence as in (a) and a VL sequence as in (b). In some embodiments,the antibody comprises a VH sequence of SEQ ID NO: 51. In someembodiments, the antibody comprises a VL sequence of SEQ ID NO: 52.

In other embodiments, the antibody comprises the following HVRs: (a) anHVR-L1 comprising the amino acid sequence of QSSQSVDENNYLS (SEQ ID NO:44); (b) an HVR-L2 comprising the amino acid sequence of RASTLES (SEQ IDNO: 45); and (c) an HVR-L3 comprising the amino acid sequence ofLGGYSGYSDDG (SEQ ID NO: 46). In some embodiments, the antibody furthercomprises the following light chain variable domain FRs: (a) FR-L1comprising the amino acid sequence of AVLTQTPSPVSAAVGGTVSISC (SEQ ID NO:47); (b) FR-L2 comprising the amino acid sequence of WFQQKPGQPPKLLIY(SEQ ID NO: 48); (c) FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTQFTLTVSGVQCDDAATYYC (SEQ ID NO: 49); and (d) FR-L4comprising the amino acid sequence of FGGGTEVVVK (SEQ ID NO: 50).

In another aspect, the invention features an isolated antibody thatspecifically binds amphiregulin, wherein the antibody comprises thefollowing HVRs: (a) an HVR-H1 comprising the amino acid sequence ofSYAIS (SEQ ID NO: 37); (b) an HVR-H2 comprising the amino acid sequenceof FIVGSSGSAYYASWAKS (SEQ ID NO: 38); (c) an HVR-H3 comprising the aminoacid sequence of GLYSGGNY (SEQ ID NO: 39); (d) an HVR-L1 comprising theamino acid sequence of QSSQSVDENNYLS (SEQ ID NO: 44); (e) an HVR-L2comprising the amino acid sequence of RASTLES (SEQ ID NO: 45); and (f)an HVR-L3 comprising the amino acid sequence of LGGYSGYSDDG (SEQ ID NO:46). In some embodiments, the antibody further comprises the followingheavy chain variable domain and light chain variable domain FRs: (a)FR-H1 comprising the amino acid sequence ofQSLEESRGGLIKPGGTLTLTCTVSGFSLS (SEQ ID NO: 40); (b) 1-'R-H2 comprisingthe amino acid sequence of WVRQAPGNGLEWIG (SEQ ID NO: 41); (c) FR-H3comprising the amino acid sequence of RSTITRDTNLNTVTLKMTSLTAADTATYFCAK(SEQ ID NO: 42); (d) FR-H4 comprising the amino acid sequence ofWGPGTLVTVSS (SEQ ID NO: 43); (e) FR-L1 comprising the amino acidsequence of AVLTQTPSPVSAAVGGTVSISC (SEQ ID NO: 47); (f) FR-L2 comprisingthe amino acid sequence of WFQQKPGQPPKLLIY (SEQ ID NO: 48); (g) FR-L3comprising the amino acid sequence of GVPSRFSGSGSGTQFTLTVSGVQCDDAATYYC(SEQ ID NO: 49); and (h) FR-L4 comprising the amino acid sequence ofFGGGTEVVVK (SEQ ID NO: 50). In some embodiments, the antibody comprisesa VH sequence of SEQ ID NO: 51 and a VL sequence of SEQ ID NO: 52.

In another aspect, the invention features an isolated antibody thatcompetes for binding to amphiregulin with any one of the precedinganti-amphiregulin antibodies.

In another aspect, the invention features an isolated antibody thatbinds to the same epitope as any one of the preceding antibodies.

In some embodiments, any one of the preceding antibodies can be amonoclonal antibody. In some embodiments, the monoclonal antibody can bea rabbit monoclonal antibody.

In some embodiments, any one of the preceding antibodies can be anantibody fragment that specifically binds human pro-epiregulin. In someembodiments, the antibody fragment is selected from the group consistingof Fab, single chain variable fragment (scFv), Fv, Fab′, Fab′-SH,F(ab′)2, and diabody.

In another aspect, the invention features an immunoconjugate comprisingany one of the preceding antibodies.

In another aspect, the invention features an isolated nucleic acid thatencodes any of the antibodies described herein. In another aspect, theinvention features a vector (e.g., an expression vector) comprising thenucleic acid for expressing the antibody. In another aspect, theinvention features host cells comprising the preceding nucleic acidsand/or vectors.

In some aspects, any one of the preceding antibodies can be for use indetecting the presence or expression level of human pro-epiregulinand/or amphiregulin in a biological sample. In some embodiments, thedetecting is by immunohistochemistry (IHC), immunofluorescence (IF), orimmunoblot. In some embodiments, the detecting is by IHC. In someembodiments, the sample comprises a fixed tissue. In some embodiments,the fixed tissue is a formalin-fixed paraffin-embedded (FFPE) tissue. Insome embodiments, the sample is from a subject having, or predisposedto, cancer or an autoimmune disease.

A further aspect of the invention is a method of detecting the presenceor expression level of human pro-epiregulin and/or amphiregulin in abiological sample comprising contacting the biological sample with anyone of the preceding antibodies and detecting the presence of the boundantibody. In some embodiments, the detecting is by IHC, IF, orimmunoblot. In some embodiments, the detecting is by IHC. In someembodiments, the sample comprises a fixed tissue. In some embodiments,the fixed tissue is a FFPE tissue. In some embodiments, the sample isfrom a subject having or predisposed to cancer or autoimmune disease.

BRIEF DESCRIPTION OF THE FIGURES

The application file contains at least one drawing executed in color.Copies of this patent or patent application with color drawings will beprovided by the Office upon request and payment of the necessary fee.

FIG. 1 is a schematic diagram showing the general antibody productionprocess for the anti-human pro-epiregulin and anti-human amphiregulinantibodies.

FIG. 2 is an image showing the results of immunohistochemistry (IHC) onformalin-fixed, paraffin-Embedded (FFPE) colon cancer tissue comparingclone J5H1L1 to a commercially available clone from Cell SignalingTechnologies, Inc.

FIG. 3 is an image of an IHC assay using two clones of anti-humanamphiregulin antibodies to stain formalin-fixed, paraffin embeddedmetastatic colorectal cancer tissue.

FIG. 4A is an image showing the results of a Western blot of EREG.

FIG. 4B is an image showing the results of a Western blot of AREG.

FIGS. 5A-5C provide representative images of IHC results for EREG andAREG IHC, demonstrating membrane, granular/punctate, and cytoplasmicstaining.

FIG. 6 is a picture of a Western blot (WB) analysis comparting EREGantibody clone J89H12L3 with clone D4O5I.

FIG. 7 demonstrates images of IHC analysis of EREG protein expression inxenograft. Samples A-D are stained with clone J89H12L3. Samples E-G arestained with clone D4O5I. Samples A and E are xenografts from SKE23cells. Samples B and F are xenografts from PLR124EREG +/− cells. SamplesC and G are xenografts from SK-Hep 1 cells. Samples D and H arexenografts from PLR124EREG −/− cells. Brown indicates positive staining.

FIG. 8 is a comparison between J89H12L3 and D4O5I in lung squamous cellcarcinoma (SCC) tissue. Images A-C are tissues stained with cloneJ89H12L3. Images D-F are tissues stained with clone D4O5I.

FIG. 9 is a comparison between J89H12L3 and D4O5I in lung adenocarcinomaand adenosquamous cell carcinoma. Images A-D are tissues stained withclone J89H12L3. Images E-H are tissues stained with clone D4O5I.

FIG. 10 is an IHC analysis of EREG protein expression in normal andtumor tissues using clone J89H12L3. Images are of tissues stained withJ89H12L3 as follows: skin squamous cell carcinoma (A), hepatocellularcarcinoma (B), bladder transitional cell carcinoma (C), colonadenocarcinoma (D), lung adenocarcinoma (E), skin (F), cervix (G), andesophagus (H).

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

The terms “anti-human pro-epiregulin antibody,” “anti-humanpro-epiregulin antibody,” “antibody that specifically binds to humanpro-epiregulin,” and “antibody that binds to human pro-epiregulin” referto an antibody that is capable of binding human pro-epiregulin withsufficient affinity such that the antibody is useful as a diagnosticand/or therapeutic agent in targeting human pro-epiregulin. In oneembodiment, the extent of binding of an anti-human pro-epiregulinantibody to an unrelated, non-human pro-epiregulin protein is less thanabout 10% of the binding of the antibody to human pro-epiregulin asmeasured, e.g., by a radioimmunoassay (RIA). In certain embodiments, anantibody that binds to human pro-epiregulin has a dissociation constant(Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM(e.g., 10⁻⁸M or less, e.g., from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to10⁻¹³ M). In certain embodiments, an anti-human pro-epiregulin antibodybinds to an epitope of human pro-epiregulin that is conserved amonghuman pro-epiregulin from different species.

The terms “anti-human amphiregulin antibody,” “anti-human amphiregulinantibody,” “antibody that specifically binds to human amphiregulin,” and“antibody that binds to human amphiregulin” refer to an antibody that iscapable of binding human amphiregulin with sufficient affinity such thatthe antibody is useful as a diagnostic and/or therapeutic agent intargeting human amphiregulin. In one embodiment, the extent of bindingof an anti-human amphiregulin antibody to an unrelated, non-humanamphiregulin protein is less than about 10% of the binding of theantibody to human amphiregulin as measured, e.g., by a radioimmunoassay(RIA). In certain embodiments, an antibody that binds to humanamphiregulin has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM,≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10⁻⁸M or less, e.g., from10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). In certainembodiments, an anti-human amphiregulin antibody binds to an epitope ofhuman amphiregulin that is conserved among human amphiregulin fromdifferent species.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isprovided herein.

An “autoimmune disease” is a disease or disorder arising from anddirected against an individual's own tissues or organs or aco-segregation or manifestation thereof or resulting conditiontherefrom. Autoimmune diseases can be an organ-specific disease (i.e.,the immune response is specifically directed against an organ systemsuch as the endocrine system, the hematopoietic system, the skin, thecardiopulmonary system, the gastrointestinal and liver systems, therenal system, the thyroid, the ears, the neuromuscular system, thecentral nervous system, etc.) or a systemic disease that can affectmultiple organ systems (for example, systemic lupus erythematosus (SLE),rheumatoid arthritis (RA), polymyositis, etc.). Non-limiting exemplaryautoimmune diseases include autoimmune rheumatologic disorders (such as,for example, RA, Sjogren's syndrome, scleroderma, lupus such as SLE andlupus nephritis, polymyositis-dermatomyositis, cryoglobulinemia,anti-phospholipid antibody syndrome, and psoriatic arthritis),autoimmune gastrointestinal and liver disorders (such as, for example,inflammatory bowel diseases {e.g., ulcerative colitis and Crohn'sdisease), autoimmune gastritis and pernicious anemia, autoimmunehepatitis, primary biliary cirrhosis, primary sclerosing cholangitis,and celiac disease), vasculitis (such as, for example, ANCA-negativevasculitis and ANCA-associated vasculitis, including Churg-Straussvasculitis, Wegener's granulomatosis, and microscopic polyangiitis),autoimmune neurological disorders (such as, for example, multiplesclerosis, opsoclonus myoclonus syndrome, myasthenia gravis,neuromyelitis optica, Parkinson's disease, Alzheimer's disease, andautoimmune polyneuropathies), renal disorders (such as, for example,glomerulonephritis, Goodpasture's syndrome, and Berger's disease),autoimmune dermatologic disorders (such as, for example, psoriasis,urticaria, hives, pemphigus vulgaris, bullous pemphigoid, and cutaneouslupus erythematosus), hematologic disorders (such as, for example,thrombocytopenic purpura, thrombotic thrombocytopenic purpura,post-transfusion purpura, and autoimmune hemolytic anemia),atherosclerosis, uveitis, autoimmune hearing diseases (such as, forexample, inner ear disease and hearing loss), Behcet's disease,Raynaud's syndrome, organ transplant, and autoimmune endocrine disorders(such as, for example, diabetic-related autoimmune diseases such asinsulin-dependent diabetes mellitus (IDDM), Addison's disease, andautoimmune thyroid disease (e.g., Graves' disease and thyroiditis)).More preferred such diseases include, for example, RA, ulcerativecolitis, ANCA-associated vasculitis, lupus, multiple sclerosis,Sjogren's syndrome, Graves' disease, IDDM, pernicious anemia,thyroiditis, and glomerulonephritis.

By “biological sample” is meant a collection of similar cells obtainedfrom a subject or patient. A biological sample can be a tissue or a cellsample. The source of the tissue or cell sample may be solid tissue asfrom a fresh, frozen and/or preserved organ or tissue sample or biopsyor aspirate; blood or any blood constituents; bodily fluids such ascerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitialfluid; cells from any time in gestation or development of the subject.The biological sample can also be obtained from in vitro tissue or cellculture. The tissue sample may contain compounds which are not naturallyintermixed with the tissue in nature such as preservatives,anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.Examples of biological samples herein include, but are not limited to,tumor biopsies, circulating tumor cells, serum or plasma, circulatingplasma proteins, ascitic fluid, primary cell cultures or cell linesderived from tumors or exhibiting tumor-like properties, as well aspreserved tumor samples, such as formalin-fixed, paraffin-embedded tumorsamples or frozen tumor samples.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include, butare not limited to, carcinoma, lymphoma (e.g., Hodgkin's andnon-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastrointestinal cancer, pancreatic cancer,glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer,hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, leukemia and other lymphoproliferative disorders, and varioustypes of head and neck cancer. In one specific embodiment, thebiological sample is a sample of a colorectal tumor. In another specificembodiment, the biological sample is a sample of a breast tumor. Inanother specific embodiment, the biological sample is a sample of a lungtumor, such as non-small cell lung carcinoma.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al. Sequences of Proteins of Immunological Interest. 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1 (L 1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.

The terms “full-length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The terms “level of expression” or “expression level” in general areused interchangeably and generally refer to the amount of apolynucleotide, mRNA, or an amino acid product or protein in abiological sample. “Expression” generally refers to the process by whichgene-encoded information is converted into the structures present andoperating in the cell. Therefore, according to the invention“expression” of a gene (e.g., the human pro-epiregulin gene) may referto transcription into a polynucleotide, translation into a protein, oreven posttranslational modification of the protein. Fragments of thetranscribed polynucleotide, the translated protein, or thepost-translationally modified protein shall also be regarded asexpressed whether they originate from a transcript generated byalternative splicing or a degraded transcript, or from apost-translational processing of the protein, e.g., by proteolysis. Insome embodiments, “expression level” refers to amount of a protein(e.g., human pro-epiregulin) in a biological sample as determined usingimmunohistochemistry (IHC), immunoblotting (e.g., Western blotting),immunofluorescence (IF), Enzyme-Linked Immunosorbant Assay (ELISA), orflow cytometry.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest. FifthEdition, NIH Publication 91-3242, Bethesda Md., Vols. 1-3, 1991. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR” as used herein refers to eachof the regions of an antibody variable domain which are hypervariable insequence (“complementarity determining regions” or “CDRs”) and/or formstructurally defined loops (“hypervariable loops”) and/or contain theantigen-contacting residues (“antigen contacts”). Generally, antibodiescomprise six HVRs: three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). Exemplary HVRs herein include:

(a) hypervariable loops occurring at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothiaet al. J. Mol. Biol. 196: 901-917, 1987);

(b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97(L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequencesof Proteins of Immunological Interest. 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md., 1991);

(c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55(L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum etal. J. Mol. Biol. 262: 732-745, 1996); and

(d) combinations of (a), (b), and/or (c), including HVR amino acidresidues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1),26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3). Unless otherwiseindicated, HVR residues and other residues in the variable domain (e.g.,FR residues) are numbered herein according to Kabat et al., supra.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al. J. Chromatogr. B. 848: 79-87, 2007.

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-human pro-epiregulin antibody”refers to one or more nucleic acid molecules encoding antibody heavy andlight chains (or fragments thereof), including such nucleic acidmolecule(s) in a single vector or separate vectors, and such nucleicacid molecule(s) present at one or more locations in a host cell.

“Isolated nucleic acid encoding an anti-human amphiregulin antibody”refers to one or more nucleic acid molecules encoding antibody heavy andlight chains (or fragments thereof), including such nucleic acidmolecule(s) in a single vector or separate vectors, and such nucleicacid molecule(s) present at one or more locations in a host cell.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, or a combination thereof.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pro-epiregulin,” as used herein, refers to any nativepro-epiregulin from any vertebrate source, including mammals such asprimates (e.g., humans) and rodents (e.g., mice and rats), unlessotherwise indicated, but does not include the cleaved and secreted form,which is referred to as “epiregulin”. The term encompasses“full-length,” unprocessed human pro-epiregulin as well as any form ofhuman pro-epiregulin that results from processing in the cell, exceptfor the cleaved and secreted form of epiregulin. The term alsoencompasses naturally occurring variants of human pro-epiregulin, e.g.,splice variants or allelic variants. The canonical pro-epiregulinmolecule is a 169 amino acid single pass type-I membrane protein that iscleaved to a secreted molecule (termed epiregulin) containing aminoacids amino acids 60-108 and which acts as a ligand of EGFR. See UniprotEntry O14944. Additional information on the human pro-epiregulin gene,including the genomic DNA sequence, can be found under NCBI Gene ID No.2069. The amino acid sequence of an exemplary full-length humanpro-epiregulin protein can be found, e.g., under NCBI Accession No.BAA22146 or UniProt Accession No. O14944, and herein at SEQ ID NO: 36.

The term “amphiregulin,” as used herein, refers to any nativeamphiregulin from any vertebrate source, including mammals such asprimates (e.g., humans) and rodents (e.g., mice and rats), unlessotherwise indicated, but does not include the cleaved and secreted form.The term encompasses “full-length,” unprocessed human amphiregulin aswell as any form of human amphiregulin that results from processing inthe cell, except for the cleaved and secreted form. The term alsoencompasses naturally occurring variants of human amphiregulin, e.g.,splice variants or allelic variants. The canonical amphiregulin moleculeis a 252 amino acid single pass type-I membrane protein that is cleavedat Lysine 187 to form a secreted EGFR ligand. See Uniprot Entry P15514;Levano and Kenny, FEBS Letters, Vol. 586, Issue 19, pp. 3500-02 (2012).Additional information on the human amphiregulin gene, including thegenomic DNA sequence, can be found under NCBI Gene ID No. 374. The aminoacid sequence of an exemplary full-length human pro-epiregulin proteincan be found, e.g., under NCBI Accession No. NP_001648 or UniProtAccession No. P15514, and herein at SEQ ID NO: 36.

As used herein, the term “specifically binds to” or is “specific for”refers to measurable and reproducible interactions such as bindingbetween a target and an antibody, which is determinative of the presenceof the target in the presence of a heterogeneous population of moleculesincluding biological molecules. For example, an antibody thatspecifically binds to a target (which can be an epitope, e.g., aminoacid residues 148-169 of a human pro-epiregulin according to SEQ ID NO:1 or amino acid residues 238-252 of a human amphiregulin according toSEQ ID NO: 36) is an antibody that binds this target with greateraffinity, avidity, more readily, and/or with greater duration than itbinds to other targets. In one embodiment, the extent of binding of anantibody to an unrelated target is less than about 10% of the binding ofthe antibody to the target as measured, e.g., by a radioimmunoassay(RIA). In certain embodiments, an antibody that specifically binds to atarget has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1nM, or ≤0.1 nM. In certain embodiments, an antibody specifically bindsto an epitope on a protein that is conserved among the protein fromdifferent species. In another embodiment, specific binding can include,but does not require exclusive binding.

A “subject” or “individual” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindtet al. Kuby Immunology. 6^(th) ed., page 91, W.H. Freeman and Co., 2007.A single VH or VL domain may be sufficient to confer antigen-bindingspecificity. Furthermore, antibodies that bind a particular antigen maybe isolated using a VH or VL domain from an antibody that binds theantigen to screen a library of complementary VL or VH domains,respectively. See, e.g., Portolano et al. J. Immunol. 150: 880-887, 1993and Clarkson et al. Nature. 352: 624-628, 1991.

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

II. Compositions and Methods

The invention provides novel antibodies that bind to humanpro-epiregulin. Antibodies of the invention are useful, for example, fordetecting the presence of human pro-epiregulin or the expression levelof human pro-epiregulin (e.g., in biological samples).

The invention also provides novel antibodies that bind to humanamphiregulin. Antibodies of the invention are useful, for example, fordetecting the presence of human amphiregulin or the expression level ofhuman amphiregulin (e.g., in biological samples).

A. Exemplary Anti-Human Pro-Epiregulin Antibodies

The invention provides anti-human pro-epiregulin antibodies useful for,e.g., diagnostic applications (e.g., immunohistochemistry (IHC),immunofluorescence (IF), and immunoblot (e.g., Western blot)). In oneexample, the invention provides anti-human pro-epiregulin antibodiesthat bind to an epitope including amino acid residues 148-169 of humanpro-epiregulin (e.g., amino acid residues 148-169 of SEQ ID NO: 1),which is located at the carboxy terminus of the pro-epiregulin molecule.In one example, the invention provides anti-human pro-epiregulinantibodies that bind to an epitope including amino acid residues 156-169of human pro-epiregulin (e.g., amino acid residues 156-169 of SEQ ID NO:1), which is located at the carboxy terminus of the pro-epiregulinmolecule. The epitope on human pro-epiregulin may be recognized in amanner that is conformation-dependent or conformation-independent.

In some instances, the anti-human pro-epiregulin antibodies that bind toamino acid residues 148-169 of human proepiregulin include at least one,two, three, four, five, or six HVRs selected from (a) HVR-H1 comprisingSEQ ID NO: 2; (b) HVR-H2 comprising SEQ ID NO: 3; (c) HVR-H3 comprisingSEQ ID NO: 4; (d) HVR-L1 comprising SEQ ID NO: 9; (e) HVR-L2 comprisingSEQ ID NO: 10; and (f) HVR-L3 comprising SEQ ID NO: 11. For example, insome instances, the anti-human pro-epiregulin antibodies include (a) anHVR-H1 comprising SEQ ID NO: 2; (b) an HVR-H2 comprising SEQ ID NO: 3;and (c) an HVR-H3 comprising SEQ ID NO: 4. In some instances, theanti-human pro-epiregulin antibodies include (a) an HVR-L1 comprisingSEQ ID NO: 9; (b) HVR-L2 comprising SEQ ID NO: 10; and (c) HVR-L3comprising SEQ ID NO: 11.

In some instances wherein the anti-human pro-epiregulin antibodies bindto amino acid residues 148-169 of human pro-epiregulin and include (a)an HVR-H1 comprising SEQ ID NO: 2; (b) an HVR-H2 comprising SEQ ID NO:3; and (c) an HVR-H3 comprising SEQ ID NO: 4, the anti-humanpro-epiregulin antibodies further include the following heavy chainvariable domain framework regions (FRs): (a) FR-H1 comprising SEQ ID NO:5; (b) FR-H2 comprising SEQ ID NO: 6; (c) FR-H3 comprising SEQ ID NO: 7;or (d) FR-H4 comprising SEQ ID NO: 8. In some instances wherein theanti-human pro-epiregulin antibodies bind to amino acid residues 148-169of human pro-epiregulin and include (a) an HVR-H1 comprising SEQ ID NO:2; (b) an HVR-H2 comprising SEQ ID NO: 3; and (c) an HVR-H3 comprisingSEQ ID NO: 4, the anti-human pro-epiregulin antibodies further includethe following heavy chain variable domain framework regions (FRs): (a)FR-H1 comprising SEQ ID NO: 5; (b) FR-H2 comprising SEQ ID NO: 6; (c)FR-H3 comprising SEQ ID NO: 7; and (d) FR-H4 comprising SEQ ID NO: 8.

In some instances wherein the anti-human pro-epiregulin antibodies bindto amino acid residues 148-169 of human proepiregulin, the antibodiesinclude (a) an HVR-H1 comprising SEQ ID NO: 2; (b) an HVR-H2 comprisingSEQ ID NO: 3; (c) an HVR-H3 comprising SEQ ID NO: 4; (d) an HVR-L1comprising SEQ ID NO: 9; (e) an HVR-L2 comprising SEQ ID NO: 10; and (f)an HVR-L3 comprising SEQ ID NO: 11. In some instances, these anti-humanpro-epiregulin antibodies include the following FRs: (a) FR-H1comprising SEQ ID NO: 5; (b) FR-H2 comprising SEQ ID NO: 6; (c) FR-H3comprising SEQ ID NO: 7; and (d) FR-H4 comprising SEQ ID NO: 8 and mayadditionally or alternatively include (e) FR-L1 comprising SEQ ID NO:12; (f) FR-L2 comprising SEQ ID NO: 13; (g) FR-L3 comprising SEQ ID NO:14; and (h) FR-L4 comprising SEQ ID NO: 15.

In some instances, the anti-human pro-epiregulin antibodies that bind toamino acid residues 148-169 of human pro-epiregulin may also include aheavy chain variable domain (VH) sequence having at least 80% (e.g., atleast 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%), at least 90%(e.g., at least 91%, 92%, 93%, or 94%), or at least 95% (e.g., at least96%, 97%, 98%, or 99%) sequence identity to, or the sequence of, theamino acid sequence of SEQ ID NO: 16. In certain embodiments, a VHsequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identitycontains substitutions (e.g., conservative substitutions), insertions,or deletions relative to the reference sequence (SEQ ID NO: 16), but ananti-human pro-epiregulin antibody including that sequence retains theability to bind to human pro-epiregulin. In certain embodiments, a totalof 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aminoacids) have been substituted, inserted, and/or deleted in SEQ ID NO: 16.In certain embodiments, substitutions, insertions, or deletions occur inregions outside the HVRs (i.e., in the FRs). Optionally, the anti-humanpro-epiregulin antibodies include the VH sequence in SEQ ID NO: 16,including post-translational modifications of that sequence. In aparticular embodiment, the VH comprises one, two, or three HVRs selectedfrom: (a) HVR-H1 comprising SEQ ID NO: 2, (b) HVR-H2 comprising SEQ IDNO: 3, and (c) HVR-H3 comprising SEQ ID NO: 4.

In some instances, the anti-human pro-epiregulin antibodies that bind toamino acid residues 148-169 of human pro-epiregulin may also include alight chain variable domain (VL) having at least 80% (e.g., at least81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%), at least 90% (e.g., atleast 91%, 92%, 93%, or 94%), or at least 95% (e.g., at least 96%, 97%,98%, or 99%) sequence identity to, or the sequence of, the amino acidsequence of SEQ ID NO: 17. In certain embodiments, a VL sequence havingat least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence (SEQ ID NO: 17), but ananti-human pro-epiregulin antibody including that sequence retains theability to bind to human pro-epiregulin. In certain embodiments, a totalof 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aminoacids) have been substituted, inserted, and/or deleted in SEQ ID NO: 17.In certain embodiments, the substitutions, insertions, or deletionsoccur in regions outside the HVRs (i.e., in the FRs). Optionally, theanti-human pro-epiregulin antibody comprises the VL sequence in SEQ IDNO: 17, including post-translational modifications of that sequence. Ina particular embodiment, the VL comprises one, two or three HVRsselected from (a) HVR-L1 comprising SEQ ID NO: 9; (b) HVR-L2 comprisingSEQ ID NO: 10; and (c) HVR-L3 comprising SEQ ID NO: 11.

In some instances, the anti-human pro-epiregulin antibodies that bind toamino acid residues 148-169 of human pro-epiregulin include both VH andVL sequences having at least 80% (e.g., at least 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, or 89%), at least 90% (e.g., at least 91%, 92%, 93%,or 94%), or at least 95% (e.g., at least 96%, 97%, 98%, or 99%) sequenceidentity to, or the sequences of, the amino acid sequences of SEQ IDNOs: 16 and 17, respectively, and may or may not includepost-translational modifications of those sequences.

In other instances, the invention provides antibodies that specificallybind human pro-epiregulin, wherein the antibodies include (a) an HVR-H1comprising SEQ ID NO: 2; (b) an HVR-H2 comprising SEQ ID NO: 3; (c) anHVR-H3 comprising SEQ ID NO: 4; (d) an HVR-L1 comprising SEQ ID NO: 9;(e) an HVR-L2 comprising SEQ ID NO: 10; and (f) an HVR-L3 comprising SEQID NO: 11. In some instances, these anti-human pro-epiregulin antibodiesinclude the following FRs: (a) FR-H1 comprising SEQ ID NO: 5; (b) FR-H2comprising SEQ ID NO: 6; (c) FR-H3 comprising SEQ ID NO: 7; and (d)FR-H4 comprising SEQ ID NO: 8 and may additionally or alternativelyinclude (e) FR-L1 comprising SEQ ID NO: 12; (f) FR-L2 comprising SEQ IDNO: 13; (g) FR-L3 comprising SEQ ID NO: 14; and (h) FR-L4 comprising SEQID NO: 15. In some embodiments, for example, the anti-humanpro-epiregulin antibodies include both a VH and a VL sequence includingthe sequences of the amino acid sequences of SEQ ID NOs: 16 and 17,respectively, and may or may not include post-translationalmodifications.

For example, the invention features anti-human pro-epiregulinantibodies, such as the anti-human pro-epiregulin antibody J5H1L1, withthe following heavy and light chain variable region sequences.

The amino acid sequence of the heavy chain variable region comprises thefollowing:

(SEQ ID NO: 16) QSVEESGGRLVTPGTPLTLTCTVSGFSLS RYGMS WVRQAPGK GLEYIGSINRTAYTYYATWAKG RFTISRTSTTVDLRMTSLTT EDTATYFCAR GLTYGGSDYDYDDALWGPGTLVTVSS

The amino acid sequence of the light chain variable region comprises thefollowing:

(SEQ ID NO: 17) QVLTQTPSSVSAAVGGTVTINC QASQSVYKNKNLA WYQQKPGQPPKL LIYRASTLAS GVSSRFKGSGSGTQFTLTISGVQCADAATYYC QGEFS CSTFDCIL FGGGTEMVVK.

In some instances, the anti-human pro-epiregulin antibodies that bind toamino acid residues 156-169 of human pro-epiregulin include at leastone, two, three, four, five, or six HVRs selected from (a) HVR-H1comprising SEQ ID NO: 18; (b) HVR-H2 comprising SEQ ID NO: 19; (c)HVR-H3 comprising SEQ ID NO: 20; (d) HVR-L1 comprising SEQ ID NO: 25 ORSEQ ID NO: 26; (e) HVR-L2 comprising SEQ ID NO: 27; and (f) HVR-L3comprising SEQ ID NO: 28. For example, in some instances, the anti-humanpro-epiregulin antibodies include (a) an HVR-H1 comprising SEQ ID NO:18; (b) an HVR-H2 comprising SEQ ID NO: 19; and (c) an HVR-H3 comprisingSEQ ID NO: 20. In some instances, the anti-human pro-epiregulinantibodies include (a) an HVR-L1 comprising SEQ ID NO: 25; (b) HVR-L2comprising SEQ ID NO: 27; and (c) HVR-L3 comprising SEQ ID NO: 28. Insome instances, the anti-human pro-epiregulin antibodies include (a) anHVR-L1 comprising SEQ ID NO: 26; (b) HVR-L2 comprising SEQ ID NO: 27;and (c) HVR-L3 comprising SEQ ID NO: 28.

In some instances wherein the anti-human pro-epiregulin antibodies bindto amino acid residues 156-169 of human pro-epiregulin and include (a)an HVR-H1 comprising SEQ ID NO: 18; (b) an HVR-H2 comprising SEQ ID NO:19; and (c) an HVR-H3 comprising SEQ ID NO: 20, the anti-humanpro-epiregulin antibodies further include the following heavy chainvariable domain framework regions (FRs): (a) FR-H1 comprising SEQ ID NO:21; (b) FR-H2 comprising SEQ ID NO: 22; (c) FR-H3 comprising SEQ ID NO:23; or (d) FR-H4 comprising SEQ ID NO: 24. In some instances wherein theanti-human pro-epiregulin antibodies bind to amino acid residues 156-169of human pro-epiregulin and include (a) an HVR-H1 comprising SEQ ID NO:18; (b) an HVR-H2 comprising SEQ ID NO: 19; and (c) an HVR-H3 comprisingSEQ ID NO: 20, the anti-human pro-epiregulin antibodies further includethe following heavy chain variable domain framework regions (FRs): (a)FR-H1 comprising SEQ ID NO: 21; (b) FR-H2 comprising SEQ ID NO: 22; (c)FR-H3 comprising SEQ ID NO: 23; and (d) FR-H4 comprising SEQ ID NO: 24.

In some instances wherein the anti-human pro-epiregulin antibodies bindto amino acid residues 156-169 of human proepiregulin, the antibodiesinclude (a) an HVR-H1 comprising SEQ ID NO: 18; (b) an HVR-H2 comprisingSEQ ID NO: 19; (c) an HVR-H3 comprising SEQ ID NO: 20; (d) an HVR-L1comprising SEQ ID NO: 25; (e) an HVR-L2 comprising SEQ ID NO: 27; and(f) an HVR-L3 comprising SEQ ID NO: 28. In some instances, theseanti-human pro-epiregulin antibodies include the following FRs: (a)FR-H1 comprising SEQ ID NO: 21; (b) FR-H2 comprising SEQ ID NO: 22; (c)FR-H3 comprising SEQ ID NO: 23; and (d) FR-H4 comprising SEQ ID NO: 24and may additionally or alternatively include (e) FR-L1 comprising SEQID NO: 29; (f) FR-L2 comprising SEQ ID NO: 30; (g) FR-L3 comprising SEQID NO: 31; and (h) FR-L4 comprising SEQ ID NO: 32.

In some instances wherein the anti-human pro-epiregulin antibodies bindto amino acid residues 156-169 of human proepiregulin, the antibodiesinclude (a) an HVR-H1 comprising SEQ ID NO: 18; (b) an HVR-H2 comprisingSEQ ID NO: 19; (c) an HVR-H3 comprising SEQ ID NO: 20; (d) an HVR-L1comprising SEQ ID NO: 26; (e) an HVR-L2 comprising SEQ ID NO: 27; and(f) an HVR-L3 comprising SEQ ID NO: 28. In some instances, theseanti-human pro-epiregulin antibodies include the following FRs: (a)FR-H1 comprising SEQ ID NO: 21; (b) FR-H2 comprising SEQ ID NO: 22; (c)FR-H3 comprising SEQ ID NO: 23; and (d) FR-H4 comprising SEQ ID NO: 24and may additionally or alternatively include (e) FR-L1 comprising SEQID NO: 29; (f) FR-L2 comprising SEQ ID NO: 30; (g) FR-L3 comprising SEQID NO: 31; and (h) FR-L4 comprising SEQ ID NO: 32.

In some instances, the anti-human pro-epiregulin antibodies that bind toamino acid residues 156-169 of human pro-epiregulin may also include aheavy chain variable domain (VH) sequence having at least 80% (e.g., atleast 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%), at least 90%(e.g., at least 91%, 92%, 93%, or 94%), or at least 95% (e.g., at least96%, 97%, 98%, or 99%) sequence identity to, or the sequence of, theamino acid sequence of SEQ ID NO: 33. In certain embodiments, a VHsequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identitycontains substitutions (e.g., conservative substitutions), insertions,or deletions relative to the reference sequence (SEQ ID NO: 33), but ananti-human pro-epiregulin antibody including that sequence retains theability to bind to human pro-epiregulin. In certain embodiments, a totalof 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aminoacids) have been substituted, inserted, and/or deleted in SEQ ID NO: 33.In certain embodiments, substitutions, insertions, or deletions occur inregions outside the HVRs (i.e., in the FRs). Optionally, the anti-humanpro-epiregulin antibodies include the VH sequence in SEQ ID NO: 33,including post-translational modifications of that sequence. In aparticular embodiment, the VH comprises one, two, or three HVRs selectedfrom: (a) HVR-H1 comprising SEQ ID NO: 18, (b) HVR-H2 comprising SEQ IDNO: 19, and (c) HVR-H3 comprising SEQ ID NO: 20.

In some instances, the anti-human pro-epiregulin antibodies that bind toamino acid residues 156-169 of human pro-epiregulin may also include alight chain variable domain (VL) having at least 80% (e.g., at least81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%), at least 90% (e.g., atleast 91%, 92%, 93%, or 94%), or at least 95% (e.g., at least 96%, 97%,98%, or 99%) sequence identity to, or the sequence of, the amino acidsequence of SEQ ID NO: 34 or SEQ ID NO: 35. In certain embodiments, a VLsequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identitycontains substitutions (e.g., conservative substitutions), insertions,or deletions relative to the reference sequence (SEQ ID NO: 34 or SEQ IDNO: 35), but an anti-human pro-epiregulin antibody including thatsequence retains the ability to bind to human pro-epiregulin. In certainembodiments, a total of 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acids) have been substituted, inserted, and/or deletedin SEQ ID NO: 34 or SEQ ID NO: 35. In certain embodiments, thesubstitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the anti-human pro-epiregulinantibody comprises the VL sequence in SEQ ID NO: 34 or SEQ ID NO: 35,including post-translational modifications of that sequence. In aparticular embodiment, the VL comprises one, two or three HVRs selectedfrom (a) HVR-L1 comprising SEQ ID NO: 25 or SEQ ID NO: 26; (b) HVR-L2comprising SEQ ID NO: 27; and (c) HVR-L3 comprising SEQ ID NO: 28.

In some instances, the anti-human pro-epiregulin antibodies that bind toamino acid residues 156-169 of human pro-epiregulin include both VH andVL sequences having at least 80% (e.g., at least 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, or 89%), at least 90% (e.g., at least 91%, 92%, 93%,or 94%), or at least 95% (e.g., at least 96%, 97%, 98%, or 99%) sequenceidentity to, or the sequences of, the amino acid sequences of SEQ IDNOs: 17 and 18, respectively, and may or may not includepost-translational modifications of those sequences.

In other instances, the invention provides antibodies that specificallybind human pro-epiregulin, wherein the antibodies include (a) an HVR-H1comprising SEQ ID NO: 18; (b) an HVR-H2 comprising SEQ ID NO: 19; (c) anHVR-H3 comprising SEQ ID NO: 20; (d) an HVR-L1 comprising SEQ ID NO: 25;(e) an HVR-L2 comprising SEQ ID NO: 27; and (f) an HVR-L3 comprising SEQID NO: 28. In some instances, these anti-human pro-epiregulin antibodiesinclude the following FRs: (a) FR-H1 comprising SEQ ID NO: 21; (b) FR-H2comprising SEQ ID NO: 22; (c) FR-H3 comprising SEQ ID NO: 23; and (d)FR-H4 comprising SEQ ID NO: 24 and may additionally or alternativelyinclude (e) FR-L1 comprising SEQ ID NO: 29; (f) FR-L2 comprising SEQ IDNO: 30; (g) FR-L3 comprising SEQ ID NO: 31; and (h) FR-L4 comprising SEQID NO: 32. In some embodiments, for example, the anti-humanpro-epiregulin antibodies include both a VH and a VL sequence includingthe sequences of the amino acid sequences of SEQ ID NOs: 17 and 18,respectively, and may or may not include post-translationalmodifications. In some embodiments, for example, the anti-humanpro-epiregulin antibodies include both a VH and a VL sequence includingthe sequences of the amino acid sequences of SEQ ID NOs: 17 and 19,respectively, and may or may not include post-translationalmodifications.

For example, the invention features anti-human pro-epiregulinantibodies, such as the anti-human pro-epiregulin antibody J89H12L3,with the following heavy and light chain variable region sequences:

The amino acid sequence of the heavy chain variable region comprises thefollowing:

(SEQ ID NO: 33) KSVEESGGRLVTPGTPLTLTCTVSGIDLS TFAMA WVRQAPGKGLEY IGFISLSDATYYATWAKG RFTISKSSSTTVDLKIITPTAEDTATYF CAR VVGDSSGYPNTFHPWGPGTLVTVSS

The amino acid sequence of the light chain variable region comprises thefollowing:

(SEQ ID NO: 34) QVLTQTPSPVSAAVGGTVTINC QASQSIHNSDFLA WYQQKPGQPPK LLIYRASKLPS GYPSRFKGSGSGTQFTLTISDLECDDAATYYC QGT YYSGGWYFT FGGGTEVVVK.

For another example, the invention features anti-human pro-epiregulinantibodies, such as the anti-human pro-epiregulin antibody J89H12L8,with the following heavy and light chain variable region sequences:

The amino acid sequence of the heavy chain variable region comprises thefollowing:

(SEQ ID NO: 33) KSVEESGGRLVTPGTPLTLTCTVSGIDLS TFAMA WVRQAPGKGLEY IGFISLSDATYYATWAKG RFTISKSSSTTVDLKIITPTAEDTATYF CAR VVGDSSGYPNTFHPWGPGTLVTVSS

The amino acid sequence of the light chain variable region comprises thefollowing:

(SEQ ID NO: 35) QVLTQTPSPVSAAVGGTVTINC QASQNIHNSDFLA WYQQKPGQPPK LLIYRASKLPS GVPSRFKGSGSGTQFTLTISDLECDDAATYYC QGT YYSGGWYFT FGGGTEVVVK.

In some instances, anti-human pro-epiregulin antibodies of the inventionare antibodies that compete for binding to human pro-epiregulin with anyone or more of the anti-human pro-epiregulin antibodies described above.In some instances, anti-human pro-epiregulin antibodies of the inventionare antibodies that bind to the same epitope or substantially the sameepitope as any one or more of the anti-human pro-epiregulin antibodiesdescribed above.

In some instances, an anti-human pro-epiregulin antibody according toany of the above embodiments may be a monoclonal antibody, comprising achimeric, humanized, or human antibody. In one embodiment, an anti-humanpro-epiregulin antibody is an antibody fragment, for example, a Fv, Fab,Fab′, scFv, diabody, or F(ab′)₂ fragment. In another embodiment, theantibody is a full-length antibody, e.g., an intact IgG antibody (e.g.,an intact IgG1 antibody) or other antibody class or isotype as definedherein.

It should be understood that the anti-human pro-epiregulin antibodies ofthe invention, although useful for the detection of the presence or theexpression level of human pro-epiregulin in a biological sample asexemplified by the Examples below, may also be used or adapted fortherapeutic use.

In further aspects, the anti-human pro-epiregulin antibodies accordingto any of the above embodiments may incorporate any of the features,singly or in combination, as described in Sections 1-5 below.

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or≤0.001 nM (e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from10⁻⁹ M to 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA) performed with the Fab version of an antibody of interestand its antigen as described by the following assay. Solution bindingaffinity of Fabs for antigen is measured by equilibrating Fab with aminimal concentration of (¹²⁵I)-labeled antigen in the presence of atitration series of unlabeled antigen, then capturing bound antigen withan anti-Fab antibody-coated plate (see, e.g., Chen et al. J. Mol. Biol.293: 865-881, 1999). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al. CancerRes. 57: 4593-4599, 1997). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20™) in PBS. When the plates have dried, 150 μl/well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmonresonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore,Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips at˜10 response units (RU). Briefly, carboxymethylated dextran biosensorchips (CM5, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (−0.2μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20(TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately25 μl/min Association rates (k_(on)) and dissociation rates (k_(off))are calculated using a simple one-to-one Langmuir binding model(BIACORE® Evaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (Kd) is calculated as the ratio k_(off)/k_(on). See, e.g., Chenet al. J. Mol. Biol. 293: 865-881, 1999. If the on-rate exceeds 10⁶ M⁻¹s⁻¹ by the surface plasmon resonance assay above, then the on-rate canbe determined by using a fluorescent quenching technique that measuresthe increase or decrease in fluorescence emission intensity(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence ofincreasing concentrations of antigen as measured in a spectrometer, suchas a stop-flow equipped spectrophometer (Aviv Instruments) or a8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with astirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)2, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9: 129-134, 2003. For a review of scFv fragments, see, e.g.,Pluckthun. The Pharmacology of Monoclonal Antibodies. Vol. 113, pp.269-315, Rosenburg and Moore eds. Springer-Verlag, New York, 1994; seealso WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. Fordiscussion of Fab and F(ab′)2 fragments comprising salvage receptorbinding epitope residues and having increased in vivo half-life, seeU.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al. Nat. Med. 9: 129-134, 2003; and Hollinger et al. Proc.Natl. Acad. Sci. USA. 90: 6444-6448, 1993. Triabodies and tetrabodiesare also described in Hudson et al. Nat. Med. 9:129-134, 2003.

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al. Proc. Natl. Acad. Sci. USA. 81:6851-6855, 1984. In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro et al. Front. Biosci. 13: 1619-1633, 2008, and are furtherdescribed, e.g., in Riechmann et al. Nature. 332: 323-329, 1988; Queenet al. Proc. Natl. Acad. Sci. USA. 86: 10029-10033, 1989; U.S. Pat. Nos.5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al. Methods.36: 25-34, 2005 (describing SDR (a-CDR) grafting); Padlan. Mol. Immunol.28: 489-498, 1991 (describing “resurfacing”); DaU' Acqua et al. Methods.36: 43-60, 2005 (describing “FR shuffling”); and Osbourn et al. Methods36: 61-68, 2005 and Klimka et al. Br. J. Cancer. 83: 252-260, 2000(describing the “guided selection” approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151: 2296, 1993); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA. 89: 4285, 1992; and Presta etal. J. Immunol. 151: 2623, 1993); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro et al. Front. Biosci. 13: 1619-1633, 2008); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al. J.Biol. Chem. 272: 10678-10684, 1997 and Rosok et al. J. Biol. Chem. 271:22611-22618, 1996).

4. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g., a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for human pro-epiregulin and the other is for any otherantigen. In certain embodiments, bispecific antibodies may bind to twodifferent epitopes of human pro-epiregulin. Bispecific antibodies mayalso be used to localize cytotoxic agents to cells which express humanpro-epiregulin. Bispecific antibodies can be prepared as full-lengthantibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein etal. Nature. 305: 537, 1983, WO 93/08829, and Traunecker et al. EMBO J.10: 3655, 1991), and “knob-in-hole” engineering (see, e.g., U.S. Pat.No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al. Science. 229: 81, 1985); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al. J. Immunol. 148(5):1547-1553, 1992); using “diabody” technology for making bispecificantibody fragments (see, e.g., Hollinger et al. Proc. Natl. Acad. Sci.USA., 90: 6444-6448, 1993); and using single-chain Fv (sFv) dimers (see,e.g. Gruber et al. J. Immunol. 152: 5368, 1994); and preparingtrispecific antibodies as described, e.g., in Tutt et al. J. Immunol.147: 60, 1991.

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to humanpro-epiregulin as well as another, different antigen (see, e.g., US2008/0069820).

5. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “preferred substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE 1 Exemplary and Preferred Amino Acid Substitutions OriginalExemplary Preferred Residue Substitutions Substitutions Ala (A) Val;Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; ArgGln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu(E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I)Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val;Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile LeuPhe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr ThrThr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser PheVal (V) Ile; Leu; Met; Phe; Ala; Norleucine LeuAmino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury. MethodsMol. Biol. 207: 179-196, 2008), and/or SDRs (a-CDRs), with the resultingvariant VH or VL being tested for binding affinity. Affinity maturationby constructing and reselecting from secondary libraries has beendescribed, e.g., in Hoogenboom et al. Methods in Molecular Biology. 178:1-37, O'Brien et al. eds., Human Press, Totowa, N.J., 2001. In someembodiments of affinity maturation, diversity is introduced into thevariable genes chosen for maturation by any of a variety of methods(e.g., error-prone PCR, chain shuffling, or oligonucleotide-directedmutagenesis). A secondary library is then created. The library is thenscreened to identify any antibody variants with the desired affinity.Another method to introduce diversity involves HVR-directed approaches,in which several HVR residues (e.g., 4-6 residues at a time) arerandomized HVR residues involved in antigen binding may be specificallyidentified, e.g., using alanine scanning mutagenesis or modeling. HVR-H3and HVR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may be outside of HVR “hotspots” orSDRs. In certain embodiments of the variant VH and VL sequences providedabove, each HVR either is unaltered, or contains no more than one, twoor three amino acid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham et al. Science. 244: 1081-1085,1989. In this method, a residue or group of target residues (e.g.,charged residues such as Arg, Asp, His, Lys, and Glu) are identified andreplaced by a neutral or negatively charged amino acid (e.g., alanine orpolyalanine) to determine whether the interaction of the antibody withantigen is affected. Further substitutions may be introduced at theamino acid locations demonstrating functional sensitivity to the initialsubstitutions. Alternatively, or additionally, a crystal structure of anantigen-antibody complex to identify contact points between the antibodyand antigen. Such contact residues and neighboring residues may betargeted or eliminated as candidates for substitution. Variants may bescreened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH. 15: 26-32, 1997. Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. Theamount of fucose is determined by calculating the average amount offucose within the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn297 (e.g., complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (EUnumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 and US2004/0093621. Examples of publications related to “defucosylated” or“fucose-deficient” antibody variants include: US 2003/0157108; WO2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol.336: 1239-1249, 2004; and Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614,2004. Examples of cell lines capable of producing defucosylatedantibodies include Lec13 CHO cells deficient in protein fucosylation(Ripka et al. Arch. Biochem. Biophys. 249: 533-545, 1986; US2003/0157108; and WO 2004/056312, especially at Example 11), andknockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8,knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614, 2004; Kanda et al. Biotechnol. Bioeng. 94(4): 680-688, 2006; andWO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878; U.S. Pat. No.6,602,684; and US 2005/0123546. Antibody variants with at least onegalactose residue in the oligosaccharide attached to the Fc region arealso provided. Such antibody variants may have improved CDC function.Such antibody variants are described, e.g., in WO 1997/30087; WO1998/58964; and WO 1999/22764.

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an anti-human pro-epiregulin antibodyof the invention (e.g., J5-H1L1) provided herein, thereby generating anFc region variant. The Fc region variant may comprise a human Fc regionsequence (e.g., a human IgG₁, IgG₂, IgG₃ or IgG₄ Fc region) comprisingan amino acid modification (e.g., a substitution) at one or more aminoacid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcyR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII andFcyRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch et al. Annu. Rev. Immunol. 9: 457-492, 1991.Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. Nos. 5,500,362 and5,821,337; Hellstrom et al. Proc. Natl. Acad. Sci. USA. 83: 7059-7063,1986; Hellstrom et al. Proc. Natl Acad. Sci. USA. 82: 1499-1502, 1985;and Bruggemann et al. J. Exp. Med. 166: 1351-1361, 1987. Alternatively,non-radioactive assays methods may be employed (see, for example, ACTI™non-radioactive cytotoxicity assay for flow cytometry (CellTechnology,Inc. Mountain View, Calif.; and CytoTox 96® non-radioactive cytotoxicityassay (Promega, Madison, Wis.). Useful effector cells for such assaysinclude peripheral blood mononuclear cells (PBMC) and Natural Killer(NK) cells. Alternatively, or additionally, ADCC activity of themolecule of interest may be assessed in vivo, e.g., in an animal modelsuch as that disclosed in Clynes et al. Proc. Natl. Acad. Sci. USA.95:652-656, 1998. C1q binding assays may also be carried out to confirmthat the antibody is unable to bind C1q and hence lacks CDC activity.See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO2005/100402. To assess complement activation, a CDC assay may beperformed (see, e.g., Gazzano-Santoro et al. J. Immunol. Methods. 202:163, 1996; Cragg et al. Blood. 101: 1045-1052, 2003; and Cragg et al.Blood 103: 2738-2743, 2004. FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art(see, e.g., Petkova et al. Intl. Immunol. 18(12): 1759-1769, 2006).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327, and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297, and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312; andShields et al. J. Biol. Chem. 9(2): 6591-6604, 2001.

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184, 2000.

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al. J. Immunol. 117: 587,1976 andKim et al, J. Immunol. 24: 249, 1994), are described in US PatentApplication No. 2005/0014934. Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).See also Duncan et al. Nature. 322:738-740, 1988; U.S. Pat. Nos.5,648,260 and 5,624,821; and WO 94/29351 concerning other examples of Fcregion variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an anti-human pro-epiregulin antibody of theinvention (e.g., J5-H1L1) provided herein may be further modified tocontain additional nonproteinaceous moieties that are known in the artand readily available. The moieties suitable for derivatization of theantibody include but are not limited to water soluble polymers.Non-limiting examples of water soluble polymers include, but are notlimited to, polyethylene glycol (PEG), copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymersor random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer isattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al. Proc. Natl. Acad. Sci. USA. 102: 11600-11605,2005). The radiation may be of any wavelength, and includes, but is notlimited to, wavelengths that do not harm ordinary cells, but which heatthe nonproteinaceous moiety to a temperature at which cells proximal tothe antibody-nonproteinaceous moiety are killed.

B. Exemplary Anti-Human Amphiregulin Antibodies

The invention provides anti-human amphiregulin antibodies useful for,e.g., diagnostic applications (e.g., immunohistochemistry (IHC),immunofluorescence (IF), and immunoblot (e.g., Western blot)). In oneexample, the invention provides anti-human amphiregulin antibodies thatbind to an epitope including amino acid residues 238-252 of humanamphiregulin (e.g., amino acid residues 238-252 of SEQ ID NO: 36), whichis located at the carboxy terminus of the amphiregulin molecule. Theepitope on human amphiregulin may be recognized in a manner that isconformation-dependent or conformation-independent.

In some instances, the anti-human amphiregulin antibodies that bind toamino acid residues 148-169 of human proepiregulin include at least one,two, three, four, five, or six HVRs selected from (a) HVR-H1 comprisingSEQ ID NO: 37; (b) HVR-H2 comprising SEQ ID NO: 38; (c) HVR-H3comprising SEQ ID NO: 39; (d) HVR-L1 comprising SEQ ID NO: 44; (e)HVR-L2 comprising SEQ ID NO: 45; and (f) HVR-L3 comprising SEQ ID NO:46. For example, in some instances, the anti-human amphiregulinantibodies include (a) an HVR-H1 comprising SEQ ID NO: 37; (b) an HVR-H2comprising SEQ ID NO: 38; and (c) an HVR-H3 comprising SEQ ID NO: 39. Insome instances, the anti-human amphiregulin antibodies include (a) anHVR-L1 comprising SEQ ID NO: 44; (b) HVR-L2 comprising SEQ ID NO: 45;and (c) HVR-L3 comprising SEQ ID NO: 46.

In some instances wherein the anti-human amphiregulin antibodies bind toamino acid residues 238-252 of human amphiregulin and include (a) anHVR-H1 comprising SEQ ID NO: 37; (b) an HVR-H2 comprising SEQ ID NO: 38;and (c) an HVR-H3 comprising SEQ ID NO: 39, the anti-human amphiregulinantibodies further include the following heavy chain variable domainframework regions (FRs): (a) FR-H1 comprising SEQ ID NO: 40; (b) FR-H2comprising SEQ ID NO: 41; (c) FR-H3 comprising SEQ ID NO: 42; or (d)FR-H4 comprising SEQ ID NO: 43. In some instances wherein the anti-humanamphiregulin antibodies bind to amino acid residues 238-252 of humanamphiregulin and include (a) an HVR-H1 comprising SEQ ID NO: 37; (b) anHVR-H2 comprising SEQ ID NO: 38; and (c) an HVR-H3 comprising SEQ ID NO:39, the anti-human amphiregulin antibodies further include the followingheavy chain variable domain framework regions (FRs): (a) FR-H1comprising SEQ ID NO: 40; (b) FR-H2 comprising SEQ ID NO: 41; (c) FR-H3comprising SEQ ID NO: 42; and (d) FR-H4 comprising SEQ ID NO: 43.

In some instances wherein the anti-human amphiregulin antibodies bind toamino acid residues 238-252 of human proepiregulin, the antibodiesinclude (a) an HVR-H1 comprising SEQ ID NO: 37; (b) an HVR-H2 comprisingSEQ ID NO: 38; (c) an HVR-H3 comprising SEQ ID NO: 39; (d) an HVR-L1comprising SEQ ID NO: 44; (e) an HVR-L2 comprising SEQ ID NO: 45; and(f) an HVR-L3 comprising SEQ ID NO: 46. In some instances, theseanti-human amphiregulin antibodies include the following FRs: (a) FR-H1comprising SEQ ID NO: 40; (b) FR-H2 comprising SEQ ID NO: 41; (c) FR-H3comprising SEQ ID NO: 42; and (d) FR-H4 comprising SEQ ID NO: 43 and mayadditionally or alternatively include (e) FR-L1 comprising SEQ ID NO:47; (f) FR-L2 comprising SEQ ID NO: 48; (g) FR-L3 comprising SEQ ID NO:49; and (h) FR-L4 comprising SEQ ID NO: 50.

In some instances, the anti-human amphiregulin antibodies that bind toamino acid residues 238-252 of human amphiregulin may also include aheavy chain variable domain (VH) sequence having at least 80% (e.g., atleast 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%), at least 90%(e.g., at least 91%, 92%, 93%, or 94%), or at least 95% (e.g., at least96%, 97%, 98%, or 99%) sequence identity to, or the sequence of, theamino acid sequence of SEQ ID NO: 51. In certain embodiments, a VHsequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identitycontains substitutions (e.g., conservative substitutions), insertions,or deletions relative to the reference sequence (SEQ ID NO: 51), but ananti-human amphiregulin antibody including that sequence retains theability to bind to human amphiregulin. In certain embodiments, a totalof 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aminoacids) have been substituted, inserted, and/or deleted in SEQ ID NO: 51.In certain embodiments, substitutions, insertions, or deletions occur inregions outside the HVRs (i.e., in the FRs). Optionally, the anti-humanamphiregulin antibodies include the VH sequence in SEQ ID NO: 51,including post-translational modifications of that sequence. In aparticular embodiment, the VH comprises one, two, or three HVRs selectedfrom: (a) HVR-H1 comprising SEQ ID NO: 37, (b) HVR-H2 comprising SEQ IDNO: 38, and (c) HVR-H3 comprising SEQ ID NO: 39.

In some instances, the anti-human amphiregulin antibodies that bind toamino acid residues 238-252 of human amphiregulin may also include alight chain variable domain (VL) having at least 80% (e.g., at least81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%), at least 90% (e.g., atleast 91%, 92%, 93%, or 94%), or at least 95% (e.g., at least 96%, 97%,98%, or 99%) sequence identity to, or the sequence of, the amino acidsequence of SEQ ID NO: 52. In certain embodiments, a VL sequence havingat least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence (SEQ ID NO: 52), but ananti-human amphiregulin antibody including that sequence retains theability to bind to human amphiregulin. In certain embodiments, a totalof 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aminoacids) have been substituted, inserted, and/or deleted in SEQ ID NO: 52.In certain embodiments, the substitutions, insertions, or deletionsoccur in regions outside the HVRs (i.e., in the FRs). Optionally, theanti-human amphiregulin antibody comprises the VL sequence in SEQ ID NO:52, including post-translational modifications of that sequence. In aparticular embodiment, the VL comprises one, two or three HVRs selectedfrom (a) HVR-L1 comprising SEQ ID NO: 44; (b) HVR-L2 comprising SEQ IDNO: 45; and (c) HVR-L3 comprising SEQ ID NO: 46.

In some instances, the anti-human amphiregulin antibodies that bind toamino acid residues 238-252 of human amphiregulin include both VH and VLsequences having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, or 89%), at least 90% (e.g., at least 91%, 92%, 93%, or94%), or at least 95% (e.g., at least 96%, 97%, 98%, or 99%) sequenceidentity to, or the sequences of, the amino acid sequences of SEQ IDNOs: 16 and 17, respectively, and may or may not includepost-translational modifications of those sequences.

In other instances, the invention provides antibodies that specificallybind human amphiregulin, wherein the antibodies include (a) an HVR-H1comprising SEQ ID NO: 37; (b) an HVR-H2 comprising SEQ ID NO: 38; (c) anHVR-H3 comprising SEQ ID NO: 39; (d) an HVR-L1 comprising SEQ ID NO: 44;(e) an HVR-L2 comprising SEQ ID NO: 45; and (f) an HVR-L3 comprising SEQID NO: 46. In some instances, these anti-human amphiregulin antibodiesinclude the following FRs: (a) FR-H1 comprising SEQ ID NO: 40; (b) FR-H2comprising SEQ ID NO: 41; (c) FR-H3 comprising SEQ ID NO: 42; and (d)FR-H4 comprising SEQ ID NO: 43 and may additionally or alternativelyinclude (e) FR-L1 comprising SEQ ID NO: 47; (f) FR-L2 comprising SEQ IDNO: 48; (g) FR-L3 comprising SEQ ID NO: 49; and (h) FR-L4 comprising SEQID NO: 50. In some embodiments, for example, the anti-human amphiregulinantibodies include both a VH and a VL sequence including the sequencesof the amino acid sequences of SEQ ID NOs: 16 and 17, respectively, andmay or may not include post-translational modifications.

For example, the invention features anti-human amphiregulin antibodies,such as the anti-human amphiregulin antibody J111H1L10, with thefollowing heavy and light chain variable region sequences.

The amino acid sequence of the heavy chain variable region comprises thefollowing:

(SEQ ID NO: 51) QSLEESRGGLIKPGGTLTLTCTVSGFSLS SYAIS WVRQAPGNGLEW IGFIVGSSGSAYYASWAKS RSTITRDTNLNTVTLKMTSLTAADTAT YFCAK GLYSGGNY WGPGTLVTVSS

The amino acid sequence of the light chain variable region comprises thefollowing:

(SEQ ID NO: 52) AVLTQTPSPVSAAVGGTVSISC QSSQSVDENNYLS WFQQKPGQPPK LLIYRASTLES GVPSRFSGSGSGTQFTLTVSGVQCDDAATYYC LGG YSGYSDDG FGGGTEVVVK.

In some instances, anti-human amphiregulin antibodies of the inventionare antibodies that compete for binding to human amphiregulin with anyone or more of the anti-human amphiregulin antibodies described above.In some instances, anti-human amphiregulin antibodies of the inventionare antibodies that bind to the same epitope or substantially the sameepitope as any one or more of the anti-human amphiregulin antibodiesdescribed above.

In some instances, an anti-human amphiregulin antibody according to anyof the above embodiments may be a monoclonal antibody, comprising achimeric, humanized, or human antibody. In one embodiment, an anti-humanamphiregulin antibody is an antibody fragment, for example, a Fv, Fab,Fab′, scFv, diabody, or F(ab′)₂ fragment. In another embodiment, theantibody is a full-length antibody, e.g., an intact IgG antibody (e.g.,an intact IgG1 antibody) or other antibody class or isotype as definedherein.

It should be understood that the anti-human amphiregulin antibodies ofthe invention, although useful for the detection of the presence or theexpression level of human amphiregulin in a biological sample asexemplified by the Examples below, may also be used or adapted fortherapeutic use.

In further aspects, the anti-human amphiregulin antibodies according toany of the above embodiments may incorporate any of the features, singlyor in combination, as described in Sections 1-5 below.

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or<0.001 nM (e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from10⁻⁹ M to 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA) performed with the Fab version of an antibody of interestand its antigen as described by the following assay. Solution bindingaffinity of Fabs for antigen is measured by equilibrating Fab with aminimal concentration of (¹²⁵I)-labeled antigen in the presence of atitration series of unlabeled antigen, then capturing bound antigen withan anti-Fab antibody-coated plate (see, e.g., Chen et al. J. Mol. Biol.293: 865-881, 1999). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al. CancerRes. 57: 4593-4599, 1997). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20™) in PBS. When the plates have dried, 150 μl/well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmonresonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore,Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips at˜10 response units (RU). Briefly, carboxymethylated dextran biosensorchips (CM5, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (−0.2μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20(TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately25 μl/min Association rates (k_(on)) and dissociation rates (k_(off))are calculated using a simple one-to-one Langmuir binding model(BIACORE® Evaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (Kd) is calculated as the ratio k_(off)/k_(on). See, e.g., Chenet al. J. Mol. Biol. 293: 865-881, 1999. If the on-rate exceeds 10⁶ M⁻¹s⁻¹ by the surface plasmon resonance assay above, then the on-rate canbe determined by using a fluorescent quenching technique that measuresthe increase or decrease in fluorescence emission intensity(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence ofincreasing concentrations of antigen as measured in a spectrometer, suchas a stop-flow equipped spectrophometer (Aviv Instruments) or a8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with astirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)2, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9: 129-134, 2003. For a review of scFv fragments, see, e.g.,Pluckthun. The Pharmacology of Monoclonal Antibodies. Vol. 113, pp.269-315, Rosenburg and Moore eds. Springer-Verlag, New York, 1994; seealso WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. Fordiscussion of Fab and F(ab′)2 fragments comprising salvage receptorbinding epitope residues and having increased in vivo half-life, seeU.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al. Nat. Med. 9: 129-134, 2003; and Hollinger et al. Proc.Natl. Acad. Sci. USA. 90: 6444-6448, 1993. Triabodies and tetrabodiesare also described in Hudson et al. Nat. Med. 9:129-134, 2003.

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al. Proc. Natl. Acad. Sci. USA. 81:6851-6855, 1984. In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro et al. Front. Biosci. 13: 1619-1633, 2008, and are furtherdescribed, e.g., in Riechmann et al. Nature. 332: 323-329, 1988; Queenet al. Proc. Natl. Acad. Sci. USA. 86: 10029-10033, 1989; U.S. Pat. Nos.5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al. Methods.36: 25-34, 2005 (describing SDR (a-CDR) grafting); Padlan. Mol. Immunol.28: 489-498, 1991 (describing “resurfacing”); DaU' Acqua et al. Methods.36: 43-60, 2005 (describing “FR shuffling”); and Osbourn et al. Methods36: 61-68, 2005 and Klimka et al. Br. J. Cancer. 83: 252-260, 2000(describing the “guided selection” approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151: 2296, 1993); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA. 89: 4285, 1992; and Presta etal. J. Immunol. 151: 2623, 1993); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro et al. Front. Biosci. 13: 1619-1633, 2008); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al. J.Biol. Chem. 272: 10678-10684, 1997 and Rosok et al. J. Biol. Chem. 271:22611-22618, 1996).

4. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g., a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for human amphiregulin and the other is for any otherantigen. In certain embodiments, bispecific antibodies may bind to twodifferent epitopes of human amphiregulin. Bispecific antibodies may alsobe used to localize cytotoxic agents to cells which express humanamphiregulin. Bispecific antibodies can be prepared as full-lengthantibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein etal. Nature. 305: 537, 1983, WO 93/08829, and Traunecker et al. EMBO J.10: 3655, 1991), and “knob-in-hole” engineering (see, e.g., U.S. Pat.No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al. Science. 229: 81, 1985); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al. J. Immunol. 148(5):1547-1553, 1992); using “diabody” technology for making bispecificantibody fragments (see, e.g., Hollinger et al. Proc. Natl. Acad. Sci.USA., 90: 6444-6448, 1993); and using single-chain Fv (sFv) dimers (see,e.g. Gruber et al. J. Immunol. 152: 5368, 1994); and preparingtrispecific antibodies as described, e.g., in Tutt et al. J. Immunol.147: 60, 1991.

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to humanamphiregulin as well as another, different antigen (see, e.g., US2008/0069820).

5. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “preferred substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE 2 Exemplary and Preferred Amino Acid Substitutions OriginalExemplary Preferred Residue Substitutions Substitutions Ala (A) Val;Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; ArgGln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu(E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I)Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val;Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile LeuPhe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr ThrThr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser PheVal (V) Ile; Leu; Met; Phe; Ala; Norleucine LeuAmino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury. MethodsMol. Biol. 207: 179-196, 2008), and/or SDRs (a-CDRs), with the resultingvariant VH or VL being tested for binding affinity. Affinity maturationby constructing and reselecting from secondary libraries has beendescribed, e.g., in Hoogenboom et al. Methods in Molecular Biology. 178:1-37, O′Brien et al. eds., Human Press, Totowa, N.J., 2001. In someembodiments of affinity maturation, diversity is introduced into thevariable genes chosen for maturation by any of a variety of methods(e.g., error-prone PCR, chain shuffling, or oligonucleotide-directedmutagenesis). A secondary library is then created. The library is thenscreened to identify any antibody variants with the desired affinity.Another method to introduce diversity involves HVR-directed approaches,in which several HVR residues (e.g., 4-6 residues at a time) arerandomized HVR residues involved in antigen binding may be specificallyidentified, e.g., using alanine scanning mutagenesis or modeling. HVR-H3and HVR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may be outside of HVR “hotspots” orSDRs. In certain embodiments of the variant VH and VL sequences providedabove, each HVR either is unaltered, or contains no more than one, twoor three amino acid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham et al. Science. 244: 1081-1085,1989. In this method, a residue or group of target residues (e.g.,charged residues such as Arg, Asp, His, Lys, and Glu) are identified andreplaced by a neutral or negatively charged amino acid (e.g., alanine orpolyalanine) to determine whether the interaction of the antibody withantigen is affected. Further substitutions may be introduced at theamino acid locations demonstrating functional sensitivity to the initialsubstitutions. Alternatively, or additionally, a crystal structure of anantigen-antibody complex to identify contact points between the antibodyand antigen. Such contact residues and neighboring residues may betargeted or eliminated as candidates for substitution. Variants may bescreened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH. 15: 26-32, 1997. Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. Theamount of fucose is determined by calculating the average amount offucose within the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn297 (e.g., complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example Asn297 refers to the asparagineresidue located at about position 297 in the Fc region (EU numbering ofFc region residues); however, Asn297 may also be located about ±3 aminoacids upstream or downstream of position 297, i.e., between positions294 and 300, due to minor sequence variations in antibodies. Suchfucosylation variants may have improved ADCC function. See, e.g., USPatent Publication Nos. US 2003/0157108 and US 2004/0093621. Examples ofpublications related to “defucosylated” or “fucose-deficient” antibodyvariants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742;WO2002/031140; Okazaki et al. J. Mol. Biol. 336: 1239-1249, 2004; andYamane-Ohnuki et al. Biotech. Bioeng. 87: 614, 2004. Examples of celllines capable of producing defucosylated antibodies include Lec13 CHOcells deficient in protein fucosylation (Ripka et al. Arch. Biochem.Biophys. 249: 533-545, 1986; US 2003/0157108; and WO 2004/056312,especially at Example 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g.,Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614, 2004; Kanda et al.Biotechnol. Bioeng. 94(4): 680-688, 2006; and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878; U.S. Pat. No.6,602,684; and US 2005/0123546. Antibody variants with at least onegalactose residue in the oligosaccharide attached to the Fc region arealso provided. Such antibody variants may have improved CDC function.Such antibody variants are described, e.g., in WO 1997/30087; WO1998/58964; and WO 1999/22764.

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an anti-human amphiregulin antibody ofthe invention (e.g., J111H1L10) provided herein, thereby generating anFc region variant. The Fc region variant may comprise a human Fc regionsequence (e.g., a human IgG₁, IgG₂, IgG₃ or IgG₄ Fc region) comprisingan amino acid modification (e.g., a substitution) at one or more aminoacid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcyR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII andFcyRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch et al. Annu. Rev. Immunol. 9: 457-492, 1991.Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. Nos. 5,500,362 and5,821,337; Hellstrom et al. Proc. Natl. Acad. Sci. USA. 83: 7059-7063,1986; Hellstrom et al. Proc. Natl Acad. Sci. USA. 82: 1499-1502, 1985;and Bruggemann et al. J. Exp. Med. 166: 1351-1361, 1987. Alternatively,non-radioactive assays methods may be employed (see, for example, ACTI™non-radioactive cytotoxicity assay for flow cytometry (CellTechnology,Inc. Mountain View, Calif.; and CytoTox 96® non-radioactive cytotoxicityassay (Promega, Madison, Wis.). Useful effector cells for such assaysinclude peripheral blood mononuclear cells (PBMC) and Natural Killer(NK) cells. Alternatively, or additionally, ADCC activity of themolecule of interest may be assessed in vivo, e.g., in an animal modelsuch as that disclosed in Clynes et al. Proc. Natl. Acad. Sci. USA.95:652-656, 1998. C1q binding assays may also be carried out to confirmthat the antibody is unable to bind C1q and hence lacks CDC activity.See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO2005/100402. To assess complement activation, a CDC assay may beperformed (see, e.g., Gazzano-Santoro et al. J. Immunol. Methods. 202:163, 1996; Cragg et al. Blood. 101: 1045-1052, 2003; and Cragg et al.Blood 103: 2738-2743, 2004. FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art(see, e.g., Petkova et al. Intl. Immunol. 18(12): 1759-1769, 2006).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327, and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297, and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312; andShields et al. J. Biol. Chem. 9(2): 6591-6604, 2001.

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184, 2000.

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al. J. Immunol. 117: 587,1976 andKim et al, J. Immunol. 24: 249, 1994), are described in US PatentApplication No. 2005/0014934. Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).See also Duncan et al. Nature. 322:738-740, 1988; U.S. Pat. Nos.5,648,260 and 5,624,821; and WO 94/29351 concerning other examples of Fcregion variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an anti-human amphiregulin antibody of theinvention (e.g., J111H1L10) provided herein may be further modified tocontain additional nonproteinaceous moieties that are known in the artand readily available. The moieties suitable for derivatization of theantibody include but are not limited to water soluble polymers.Non-limiting examples of water soluble polymers include, but are notlimited to, polyethylene glycol (PEG), copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymersor random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer isattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al. Proc. Natl. Acad. Sci. USA. 102: 11600-11605,2005). The radiation may be of any wavelength, and includes, but is notlimited to, wavelengths that do not harm ordinary cells, but which heatthe nonproteinaceous moiety to a temperature at which cells proximal tothe antibody-nonproteinaceous moiety are killed.

C. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-human pro-epiregulin antibodydescribed herein (e.g., J5-H1L1, J89H12L3, and J89H12L8) or ananti-human amphiregulin (e.g. J111-H1L10) is provided. Such nucleic acidmay encode an amino acid sequence comprising the VL and/or an amino acidsequence comprising the VH of the antibody (e.g., the light and/or heavychains of the antibody). In a further embodiment, one or more vectors(e.g., expression vectors) comprising such nucleic acid are provided. Ina further embodiment, a host cell comprising such nucleic acid isprovided. In one such embodiment, a host cell comprises (e.g., has beentransformed with): (1) a vector comprising a nucleic acid that encodesan amino acid sequence comprising the VL of the antibody and an aminoacid sequence comprising the VH of the antibody, or (2) a first vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VL of the antibody and a second vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VH of the antibody.In one embodiment, the host cell is eukaryotic, e.g. a Chinese HamsterOvary (CHO) cell or lymphoid cell (e.g., Y0, NSO, Sp20 cell). In oneembodiment, a method of making an anti-human pro-epiregulin antibody isprovided, wherein the method comprises culturing a host cell comprisinga nucleic acid encoding the antibody, as provided above, underconditions suitable for expression of the antibody, and optionallyrecovering the antibody from the host cell (or host cell culturemedium).

For recombinant production of an anti-human pro-epiregulin antibodydescribed herein (e.g., J5-H1L1, J89H12L3, and J89H12L8) or ananti-human amphiregulin (e.g. J111-H1L10), nucleic acid encoding anantibody, e.g., as described above, is isolated and inserted into one ormore vectors for further cloning and/or expression in a host cell. Suchnucleic acid may be readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains of theantibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. See also Charlton. Methods inMolecular Biology. Vol. 248, pp. 245-254, B. K. C. Lo, ed., HumanaPress, Totowa, N.J., 2003, describing expression of antibody fragmentsin E. coli. After expression, the antibody may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross. Nat. Biotech. 22: 1409-1414, 2004 and Li et al.Nat. Biotech. 24: 210-215, 2006.

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al. J. Gen Virol. 36:59, 1977);baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells asdescribed, e.g., in Mather. Biol. Reprod. 23:243-251, 1980); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3 A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al. Annals N.Y. Acad. Sci. 383:44-68, 1982; MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR″ CHO cells (Urlaub et al.Proc. Natl. Acad. Sci. USA. 77: 4216, 1980); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki et al. Methods inMolecular Biology. Vol. 248, pp. 255-268, B. K. C. Lo, ed., HumanaPress, Totowa, N.J., 2003.

D. Assays

Anti-human pro-epiregulin antibodies provided herein may be identified,screened for, or characterized for their physical/chemical propertiesand/or biological activities by various assays known in the art.

1. Binding assays and other assays

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, Western blot,immunohistochemistry, immunofluorescence, etc.

In another aspect, competition assays may be used to identify anantibody that competes with any one of the antibodies of the inventionfor binding to human pro-epiregulin (e.g., anti-human pro-epiregulinantibody J5H1L1, J89H12L3, and J89H12L8) or to amphiregulin (e.g.anti-human amphiregulin antibody J111H1L10). In certain embodiments,such a competing antibody binds to the same epitope (e.g., a linear or aconformational epitope) that is bound by any one of the antibodies ofthe invention (e.g., anti-human pro-epiregulin antibody J5H1L1,J89H12L3, or J89H12L8 or anti-human amphiregulin antibody J111H1L10).Detailed exemplary methods for mapping an epitope to which an antibodybinds are provided in Morris “Epitope Mapping Protocols,” in Methods inMolecular Biology Vol. 66 (Humana Press, Totowa, N.J., 1996).

In an exemplary competition assay, immobilized human pro-epiregulin isincubated in a solution comprising a first labeled antibody that bindsto human pro-epiregulin (e.g., anti-human pro-epiregulin antibodyJ5H1L1, J89H12L3, and J89H12L8) and a second unlabeled antibody that isbeing tested for its ability to compete with the first antibody forbinding to human pro-epiregulin. The second antibody may be present in ahybridoma supernatant. As a control, immobilized human pro-epiregulin isincubated in a solution comprising the first labeled antibody but notthe second unlabeled antibody. After incubation under conditionspermissive for binding of the first antibody to human pro-epiregulin,excess unbound antibody is removed, and the amount of label associatedwith immobilized human pro-epiregulin is measured. If the amount oflabel associated with immobilized human pro-epiregulin is substantiallyreduced in the test sample relative to the control sample, then thatindicates that the second antibody is competing with the first antibodyfor binding to human pro-epiregulin. See, e.g., Harlow et al.Antibodies: A Laboratory Manual. Ch. 14 (Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1988).

In another exemplary competition assay, immobilized human amphiregulinis incubated in a solution comprising a first labeled antibody thatbinds to human amphiregulin (e.g., anti-human amphiregulin antibodyJ111H1L10) and a second unlabeled antibody that is being tested for itsability to compete with the first antibody for binding to humanamphiregulin. The second antibody may be present in a hybridomasupernatant. As a control, immobilized human amphiregulin is incubatedin a solution comprising the first labeled antibody but not the secondunlabeled antibody. After incubation under conditions permissive forbinding of the first antibody to human amphiregulin, excess unboundantibody is removed, and the amount of label associated with immobilizedhuman amphiregulin is measured. If the amount of label associated withimmobilized human amphiregulin is substantially reduced in the testsample relative to the control sample, then that indicates that thesecond antibody is competing with the first antibody for binding tohuman amphiregulin. See, e.g., Harlow et al. Antibodies: A LaboratoryManual. Ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,1988).

2. Detection Assays

In one aspect, assays are provided for identifying anti-humanpro-epiregulin antibodies useful for detecting the presence of humanpro-epiregulin, e.g., in immunohistochemistry (IHC) orimmunofluorescence (IF) assays. In certain embodiments, an antibody ofthe invention is tested for such activity.

E. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-humanpro-epiregulin antibody herein conjugated to one or more labels and/oragents, such as radioactive isotopes.

In one embodiment, an immunoconjugate comprises an antibody as describedherein conjugated to a radioactive atom to form a radioconjugate. Avariety of radioactive isotopes are available for the production ofradioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸,Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu. When theradioconjugate is used for detection, it may comprise a radioactive atomfor scintigraphic studies, for example tc99m or I123, or a spin labelfor nuclear magnetic resonance (NMR) imaging (also known as magneticresonance imaging, MRI), such as iodine-123 again, iodine-131,indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium,manganese or iron.

Conjugates of an anti-human pro-epiregulin antibody and label or agentmay be made using a variety of bifunctional protein coupling agents suchas N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the label oragent. For example, an acid-labile linker, peptidase-sensitive linker,photolabile linker, dimethyl linker or disulfide-containing linker(Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020)may be used.

The immunuoconjugates herein expressly contemplate, but are not limitedto such conjugates prepared with cross-linker reagents including, butnot limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA,SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS,sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

F. Methods and Compositions for Diagnostics and Detection

In certain embodiments, the anti-human pro-epiregulin antibodiesprovided herein are useful for detecting the presence of humanpro-epiregulin in a biological sample. Likewise, the anti-humanamphiregulin antibodies provided herein are useful for detecting thepresence of human amphiregulin in a biological sample. The term“detecting” as used herein encompasses quantitative or qualitativedetection.

In one instance, an anti-human pro-epiregulin antibody (e.g., J5-H1L1,J89H12L3, or J89H12L8) for use in a method of diagnosis or detection isprovided. In one instance, for example, a method of detecting thepresence of human pro-epiregulin in a biological sample, describedbelow, is provided. In certain embodiments, the method comprisescontacting the biological sample with an anti-human pro-epiregulinantibody as described herein under conditions permissive for binding ofthe anti-human pro-epiregulin antibody to human pro-epiregulin, anddetecting whether a complex is formed between the anti-humanpro-epiregulin antibody and human pro-epiregulin. Such method may be anin vitro or in vivo method. Anti-human pro-epiregulin antibodies of theinvention (e.g., J5-H1L1, J89H12L3, or J89H12L8) can be used, forexample, in immunoassays, including, for example, immunohistochemistry(IHC), immunofluorescence (IF), immunoblotting (e.g., Western blotting),flow cytometry, and Enzyme-linked Immunosorbant Assay (ELISA). In oneembodiment, an anti-human pro-epiregulin antibody is used to selectsubjects eligible for therapy with an anti-human pro-epiregulinantibody, for example, where human pro-epiregulin is a biomarker forselection of patients.

In one instance, an anti-human amphiregulin antibody (e.g., J111H1L10)for use in a method of diagnosis or detection is provided. In oneinstance, for example, a method of detecting the presence of humanamphiregulin in a biological sample, described below, is provided. Incertain embodiments, the method comprises contacting the biologicalsample with an anti-human amphiregulin antibody as described hereinunder conditions permissive for binding of the anti-human amphiregulinantibody to human amphiregulin, and detecting whether a complex isformed between the anti-human amphiregulin antibody and humanamphiregulin. Such method may be an in vitro or in vivo method.Anti-human amphiregulin antibodies of the invention (e.g., J111H1L10)can be used, for example, in immunoassays, including, for example,immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting(e.g., Western blotting), flow cytometry, and Enzyme-linkedImmunosorbant Assay (ELISA). In one embodiment, an anti-humanamphiregulin antibody is used to select subjects eligible for therapywith an anti-human amphiregulin antibody, for example, where humanamphiregulin is a biomarker for selection of patients.

In certain instances, labeled anti-human pro-epiregulin and/oranti-human amphiregulin antibodies are provided. Labels include, but arenot limited to, labels or moieties that are detected directly (such asfluorescent, chromophoric, electron-dense, chemiluminescent, andradioactive labels), as well as moieties, such as enzymes or ligands,that are detected indirectly, for example, through an enzymatic reactionor molecular interaction. Exemplary labels include, but are not limitedto, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I, fluorophores such asrare earth chelates or fluorescein and its derivatives, rhodamine andits derivatives, dansyl, umbelliferone, luceriferases, e.g., fireflyluciferase and bacterial luciferase (U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme,saccharide oxidases, e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricaseand xanthine oxidase, coupled with an enzyme that employs hydrogenperoxide to oxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase, biotin/avidin, spin labels, bacteriophage labels,stable free radicals, and the like.

It is also understood that any of the above methods for diagnosis and/ordetection may be carried out using an immunoconjugate of the invention,as described above, in place of or in addition to an unconjugatedanti-human pro-epiregulin or anti-human amphiregulin antibody.

G. Biological Samples

In certain embodiments, the anti-human pro-epiregulin and/or anti-humanamphiregulin antibodies of the invention (e.g., J5H1L1, J89H12L3,J89H12L8 and/or J111H1L10) can be used to detect the presence of humanpro-epiregulin and/or human amphiregulin in biological samples usingmethods known in the art or described herein.

In some instances a biological sample includes a tissue or a cellsample. For example, a biological sample may include a cell or tissuefrom normal or cancer patients, such as, for example, normal andcancerous tissue of breast, colon, lung, kidney, bone, brain, muscle,stomach, pancreas, bladder, ovary, uterus, as well as heart, embryonic,and placental tissue.

In certain instances the source of the tissue or cell sample may besolid tissue as from a fresh, frozen and/or preserved organ or tissuesample or biopsy or aspirate; blood or any blood constituents; bodilyfluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid,or interstitial fluid; cells from any time in gestation or developmentof the subject. In some embodiments the biological sample is obtainedfrom in vitro tissue or cell culture. Examples of biological samplesherein include, but are not limited to, tumor biopsies, circulatingtumor cells, serum or plasma, circulating plasma proteins, asciticfluid, primary cell cultures or cell lines derived from tumors orexhibiting tumor-like properties, as well as preserved tumor samples,such as formalin-fixed, paraffin-embedded (FFPE) tumor samples or frozentumor samples.

In some embodiments the biological sample contains compounds which arenot naturally intermixed with the tissue in nature such aspreservatives, anticoagulants, buffers, nutrients, antibiotics, or thelike. In certain embodiments the biological sample has been exposed toand/or contains one or more fixatives. Fixatives that can be used withmethods and compositions of the invention include formalin,glutaraldehyde, osmium tetraoxide, acetic acid, ethanol, acetone, picricacid, chloroform, potassium dichromate and mercuric chloride and/orstabilizing by microwave heating or freezing.

In some embodiments, the biological sample is from a subject having,predisposed to, or being tested for an autoimmune disease. In certainembodiments, the autoimmune disease is an autoimmune rheumatologicdisorder (including rheumatoid arthritis, Sjogren's syndrome,scleroderma, lupus such as SLE and lupus nephritis,polymyositis-dermatomyositis, cryoglobulinemia, anti-phospholipidantibody syndrome, and psoriatic arthritis), an autoimmunegastrointestinal and liver disorder (including inflammatory boweldiseases (e.g., ulcerative colitis and Crohn's disease), autoimmunegastritis and pernicious anemia, autoimmune hepatitis, primary biliarycirrhosis, primary sclerosing cholangitis, and celiac disease),vasculitis (including ANCA-negative vasculitis and ANCA-associatedvasculitis, including Churg-Strauss vasculitis, Wegener'sgranulomatosis, and microscopic polyangiitis), an autoimmuneneurological disorder (including multiple sclerosis, opsoclonusmyoclonus syndrome, myasthenia gravis, neuromyelitis optica, Parkinson'sdisease, Alzheimer's disease, and autoimmune polyneuropathies), a renaldisorder (including glomerulonephritis, Goodpasture's syndrome, andBerger's disease), an autoimmune dermatologic disorder (includingpsoriasis, urticaria, hives, pemphigus vulgaris, bullous pemphigoid, andcutaneous lupus erythematosus), a hematologic disorder (includingthrombocytopenic purpura, thrombotic thrombocytopenic purpura,post-transfusion purpura, and autoimmune hemolytic anemia),atherosclerosis, uveitis, an autoimmune hearing disease (including innerear disease and hearing loss), Behcet's disease, Raynaud's syndrome,organ transplant, or an autoimmune endocrine disorder (includingdiabetic-related autoimmune diseases such as insulin-dependent diabetesmellitus (IDDM), Addison's disease, and autoimmune thyroid disease(including Graves' disease and thyroiditis)).

In other embodiments, the biological sample is from a subject having,predisposed to, or being tested for cancer. In certain embodiments thecancer is carcinoma, lymphoma (including Hodgkin's and non-Hodgkin'slymphoma), blastoma, sarcoma, leukemia, squamous cell cancer, small-celllung cancer, non-small cell lung cancer, adenocarcinoma of the lung,squamous carcinoma of the lung, cancer of the peritoneum, hepatocellularcancer, gastrointestinal cancer, pancreatic cancer, glioma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney cancer, liver cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma,leukemia and other lymphoproliferative disorders, or various types ofhead and neck cancer. In one specific example, the biological sample isa colorectal tumor sample

III. Examples

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Rabbit monoclonal antibodies were raised against synthetic peptidescorresponding to the C-terminus of human precursor EREG/AREG proteins.As a result, these antibodies detect the membrane-bound forms ofpro-EREG and pro-AREG proteins prior to their cleavage but also detectthe cytosolic fragment of the precursor proteins prior to beingtransported to the cell membrane.

Example 1 Generation of Anti-Human Pro-Epiregulin Antibodies AgainstAmino Acids 148-169 of SEQ ID NO: 1

Anti-human pro-epiregulin rabbit monoclonal antibodies were generated asschematically depicted in FIG. 1 .

Briefly, the peptide fragment of amino acid residues 148-169 of SEQ IDNO: I was synthesized and conjugated via glutaraldehyde to keyholelimpet hemocyanin (KLH), an extensively used carrier protein forstimulating a substantial immune response via antibody production. NewZealand. White rabbits were immunized with KLH conjugated humanpro-epiregulin antigen emulsified with complete Freund's adjuvantfollowed by a series of human pro-epiregulin antigen booster emulsifiedwith incomplete Freund's adjuvant. The antibody-expressing cells werescreened by enzyme-linked immunoabsorbant assay (ELISA) using the humanpro-epiregulin antigen. All ELISA positive clones were further screenedby immunohistochemistry (IHC), and the clone producing the antibody withthe highest specificity was selected. For recombinant production ofanti-human pro-epiregulin antibodies, cDNA coding for the heavy chainand light chain sequences of the antibodies were cloned, expressed byco-transfection, and screened for binding to human pro-epiregulin byIHC. Anti-human pro-epiregulin monoclonal antibody J5H1L1 was producedusing these methods and subsequently purified by Protein A affinitychromatography. The heavy and light chain variable region sequences ofthe J5-H1L1 antibody are as follows.

The amino acid sequence of the heavy chain variable region comprises thefollowing:

(SEQ ID NO: 16) QSVEESGGRLVTPGTPLTLTCTVSGFSLSRYGMSWVRQAPGKGLEYIGSINRTAYTYYATWAKGRFTISRTSTTVDLRMTSLTTEDTATYFCARGLTYGGSDYDYDDALWGPGTLVTVSS

The amino acid sequence of the light chain variable region comprises thefollowing:

(SEQ ID NO: 17) QVLTQTPSSVSAAVGGTVTINCQASQSVYKNKNLAWYQQKPGQPPKLLIYRASTLASGVSSRFKGSGSGTQFTLTISGVQCADAATYYCQGE FSCSTFDCILFGGGTEMVVK.

Example 2 Generation of Anti-Human Amphiregulin Antibodies

Anti-human amphiregulin rabbit monoclonal antibodies were generated asschematically depicted in FIG. 1 . Briefly, the peptide fragment ofamino acid residues 238-252 of SEQ ID NO: 36 was synthesized andconjugated via glutaraldehyde to keyhole limpet hemocyanin (KLH), anextensively used carrier protein for stimulating a substantial immuneresponse via antibody production. New Zealand White rabbits wereimmunized with KLH conjugated human amphiregulin antigen emulsified withcomplete Freund's adjuvant followed by a series of human amphiregulinantigen booster emulsified with incomplete Freund's adjuvant. Theantibody-expressing cells were screened by enzyme-linked immunoabsorbantassay (ELISA) using the human amphiregulin antigen. All ELISA positiveclones were further screened by immunohistochemistry (IHC), and theclone producing the antibody with the highest specificity was selected.For recombinant production of anti-human amphiregulin antibodies, cDNAcoding for the heavy chain and light chain sequences of the antibodieswere cloned, expressed by co-transfection, and screened for binding tohuman amphiregulin by IHC. Anti-human amphiregulin monoclonal antibodyJ111H1L10 was produced using these methods and subsequently purified byProtein A affinity chromatography. The heavy and light chain variableregion sequences of the J111H1L10 antibody are as follows.

The amino acid sequence of the heavy chain variable region comprises thefollowing:

(SEQ ID NO: 51) QSLEESRGGLIKPGGTLTLTCTVSGFSLSSYAISWVRQAPGNGLEWIGFIVGSSGSAYYASWAKSRSTITRDTNLNTVTLKMTSLTAADTAT YFCAKGLYSGGNYWGPGTLVTVSS

The amino acid sequence of the light chain variable region comprises thefollowing:

(SEQ ID NO: 52) AVLTQTPSPVSAAVGGTVSISCQSSQSVDENNYLSWFQQKPGQPPKLLIYRASTLESGVPSRFSGSGSGTQFTLTVSGVQCDDAATYYCLGG YSGYSDDGFGGGTEVVVK.

Example 3 Immunohistochemical Assays Using Anti-Human Pro-Epiregulin andAnti-Human Amphiregulin Antibodies

Reagents

All assay optimization were performed on human non-small cell lungcarcinoma (NSCLC) (NCI-H1975; NCI-H1650; H460; NCI-HCC827; H2228;Calu-3; H820), breast (MCF7; A431) carcinoma cell lines and colorectalcancer tissues. Stained slides for the tested conditions were evaluatedby a trained pathologist and the optimal staining conditions weredetermined based on the staining intensity, percentage of positive cellsand the level of non-specific (background) staining. The examined andthe nominal conditions (bold) developed for EREG/AREG IHC is shown inTable 3.

TABLE 3 The examined and the nominal assay conditions (bold) forEREG/AREG IHC assays Procedure XT OptiView DAB IHC Antibody EREG EREGEREG EREG EREG AREG AREG D4O5I S17H9L6 J59H1L2 J23H3.12L7 J5H1L1 J13H2L6J111H1L10 Sample Type Carcinoma cell lines; Colorectal cancer casesParaffin Selected De- Selected paraffinization Cell CC1: 32 CC1: 0-32-CC1: 0-32- CC1: 32-64- CC1: 16-32- CC1: 0-32- CC1: 32-48- Conditioningmin 64-92 min 64-92 min 92 min 48-64- 64-92 min 64-80 min CC2: 8-16-CC2: 8-16- 92 min CC2: 8-16- 32 min 32 min 32 min Peroxidase Pre- Pre-Pre- Post- Post- Pre- Pre- inhibition peroxidase peroxidase peroxidaseperoxidase peroxidase peroxidase peroxidase selected selected selectedselected selected selected selected Primary Ab 2 0.5; 1; 5; 0.1; 0.5; 1;0.1; 0.25; 0.5-1-2.5- 0.1; 0.5; 1; 0.1; 0.5; 1; Concentration 7.5; 10;15; 2.5; 5; 10; 0.5; 1; 2.5; 3-5-10- 2; 3; 4; 5; 2; 5; 10; (μg/ml) 20;25; 30; 20; 25; 50; 5; 10 25-50 7.5; 10; 25; 50 50; 100 100 Ab Diluent95028 90039 90040 95119 95119 90103 95119 Primary Ab 37° C. No heat; Noheat; No heat; No heat; No heat; No heat; Temperature 37° C.; 42° C. 37°C.; 42° C. 37° C.; 42° C. 37° C.; 42° C. 37° C.; 42° C. 37° C.; 42° C.Primary Ab 16 min 8-12-16- 16 min 16-20-24 16-20-24 8-16-20- 8-12-16Incubation 20-24 min min min 24-28-32- min 60 Amplify Not Selected NotNot Not Not Not Selected (4/4) selected Selected Selected SelectedSelected Counterstain Hematoxylin II, 4 min Bluing reagent, 4 minStaining Good Less Less Good The best Good The best Result stainingsensitive; sensitive sensitivity quality staining quality non- non- buthigh staining but low staining specific specific non-specificsensitivity staining in staining in staining in stroma and nucleus;testis normal stroma and colon normal colon

Of the six anti-EREG rabbit monoclonal antibodies, clone D4O5I obtainedfrom CST and clone J5H1L1 developed by Spring Bioscience providedacceptable staining. Although both EREG clones showed similarspecificity, anti-EREG clone J5H1L1 was more sensitive (FIG. 2 ). EREGprotein expression was mainly found in tumor cells but clone J5H1L1 wasable to detect basal level of expression in normal colonic epithelium aswell.

Similar to EREG IHC, AREG immunohistochemistry showed varying levels ofprotein expression in tumor cells and weak expression in normal colon.Of the two AREG rabbit monoclonal antibodies, clone J111H1L10 performedbetter by being more sensitive and more specific compared to cloneJ13H2L6. FIG. 3 represents images for the AREG IHC staining in coloncancers.

Specificity of EREG/AREG antibodies was tested by Western-blot analysisof extracts from various control cell lines. FIGS. 4A and 4B show thatboth EREG/AREG rabbit monoclonal antibodies are specific and recognizethe unglycosylated form and the C-terminal fragment of the precursorproteins.

The assay performance of EREG/AREG IHC assays were evaluated on 8colorectal cancer cases kindly provided by Dr. Paul Waring. Specimenswere immunohistochemically stained for EREG expression with clone J5H1L1and for AREG expression with J111H1L10 on a VENTANA BenchMark XT IHC/ISHinstrument. The following instrument protocols were used:

TABLE 4 EREG AREG 1. Paraffin [Selected]; 1. Paraffin [Selected]; 2.Deparaffinization [Selected]; 2. Deparaffinization [Selected]; 3. CellConditioning [Selected]; 3. Cell Conditioning [Selected]; 4. CC1[Selected]; 4. CC1 [Selected]; 5. CC1 8 Min [Selected]; 5. CC1 8 Min[Selected]; 6. CC1 16 Min [Selected]; 6. CC1 16 Min [Selected]; 7. CC124 Min [Selected]; 7. CC1 24 Min [Selected]; 8. CC1 32 Min [Selected];8. CC1 32 Min [Selected]; 9. CC1 40 Min [Selected]; 9. CC1 40 Min[Selected]; 10. CC1 48 Min [Selected]; 10. CC1 48 Min [Selected]; 11.CC1 56 Min [Selected]; 11. CC1 56 Min [Selected]; 12. CC1 64 Min[Selected]; 12. CC1 64 Min [Selected]; 13. Primary Antibody [Selected];13. Pre Primary Peroxidase Inhib. 14. Primary Antibody Temperature[Selected]; [Selected]; 14. Primary Antibody [Selected]; 15. WarmupSlide to Ab Incubation 15. Primary Antibody Temperature Temperatures[Primary Antibody] [Selected]; 16. Apply Coverslip, One Drop of 16.Warmup Slide to Ab Incubation [ANTIBODY 3] (Antibody), and Temperatures[Primary Antibody] Incubate for [0 Hr 16 Min]; 17. Apply Coverslip, OneDrop of 17. Post Primary Peroxidase Inhib. [ANTIBODY 81] (Antibody), and[Selected]; Incubate for [0 Hr 12 Min]; 18. Counterstain [Selected]; 18.Counterstain [Selected]; 19. Apply one drop of [HEMATOXYLIN 19. Applyone drop of [HEMATOXYLIN II] (Counterstain), Apply Coverslip, II](Counterstain), Apply Coverslip, and Incubate for [4 Minutes] andIncubate for [4 Minutes] 20. Post Counterstain [Selected] 20. PostCounterstain [Selected] 21. Apply one drop of [BLUING 21. Apply one dropof [BLUING REAGENT] (Post Counterstain), REAGENT] (Post Counterstain),Apply Coverslip, and Incubate for [4 Apply Coverslip, and Incubate for[4 Minutes]. Minutes].

The stained samples were scored by a trained pathologist. Both IHCdetected heterogeneous EREG/AREG protein expression resulting invariable signal intensity and patterns of staining (Table 5).

TABLE 5 EREG IHC AREG IHC Staining Staining Intensity % Intensity % CASEID (0-3) Positive Comments (0-3) Positive Comments 08A-3404-E 0 0 0 0Cytoblush 08A-822-C 0 0 2 (membrane) <1 Edge effect 08A-498-D 2(membrane) 50 Cytoblush 3 (punctate/granular) 07A-10206- 1 (cytoplasmic)10 1 (cytoplasmic) 45 1B 3 (membrane) 3 (membrane) 35 09A-3848-B 2(membrane) 50 3 (punctate/granular) 07A-20580-F 3 (membrane) 10 3(membrane) 50 08A-1198-C 3 (membrane) 75 3 (punctate/granular)06A-18241-C 3 (membrane) 40 3 (membrane) 10

Three types of staining pattern were characteristic across 8 tissuesamples: cytoplasmic, membranous and granular/punctate staining (FIG.5A-5C). EREG/AREG cytoplasmic expression is likely due to the detectionof the cytosolic fragments of the precursor proteins after theircleavage. Granular/punctate staining is associated with the detection ofthe ligands incorporated into exosomes. The membranous stainingcorresponds to the identification of the membrane anchored precursorEREG/AREG proteins.

Example 4 Generation of Anti-Human Pro-Epiregulin Antibodies AgainstAmino Acids 156-169 of SEQ ID NO: 1

Rabbit monoclonal antibodies were raised against synthetic peptidescorresponding to the C-terminus of human precursor EREG proteins. As aresult, these antibodies detect the membrane-bound forms of pro-EREGprotein prior to their cleavage but also detect the cytosolic fragmentof the precursor proteins prior to being transported to the cellmembrane.

Anti-human pro-epiregulin rabbit monoclonal antibodies were generated asschematically depicted in FIG. 1 . Briefly, the peptide fragment ofamino acid residues 156-169 of SEQ ID NO: 1 (sequence YERVTSGDPELPQV,SEQ ID NO: 36) was synthesized and an additional two amino acids(Cys-Gly) were added to the N-terminus of the sequence during synthesisto facilitate conjugation to the carrier protein KLH, an extensivelyused carrier protein for stimulating a substantial immune response viaantibody production. New Zealand White rabbits were immunized with KLHconjugated human pro-epiregulin antigen emulsified with completeFreund's adjuvant followed by a series of human pro-epiregulin antigenbooster emulsified with incomplete Freund's adjuvant. Theantibody-expressing cells were screened by enzyme-linked immunoabsorbantassay (ELISA) using the human pro-epiregulin antigen. All ELISA positiveclones were further screened by immunohistochemistry (IHC), and theclone producing the antibody with the highest specificity was selected.For recombinant production of anti-human pro-epiregulin antibodies, cDNAcoding for the heavy chain and light chain sequences of the antibodieswere cloned, expressed by co-transfection, and screened for binding tohuman pro-epiregulin by IHC.

Anti-human pro-epiregulin monoclonal antibodies J89H12L3 and J89H12L8were produced using these methods and subsequently purified by Protein Aaffinity chromatography. The heavy and light chain variable regionsequences of the J89H12L3 antibody are as follows.

The amino acid sequence of the heavy chain variable region comprises thefollowing:

(SEQ ID NO: 33) KSVEESGGRLVTPGTPLTLTCTVSGIDLSTFAMAWVRQAPGKGLEYIGFISLSDATYYATWAKGRFTISKSSSTTVDLKIITPTAEDTATYFCARVVGDSSGYPNTFHPWGPGTLVTVSS

The amino acid sequence of the light chain variable region comprises thefollowing:

(SEQ ID NO: 34) QVLTQTPSPVSAAVGGTVTINCQASQSIHNSDFLAWYQQKPGQPPKLLIYRASKLPSGVPSRFKGSGSGTQFTLTISDLECDDAATYYCQGT YYSGGWYFTFGGGTEVVVK.The heavy and light chain variable region sequences of the J89H12L8antibody are as follows.

The amino acid sequence of the heavy chain variable region comprises thefollowing:

(SEQ ID NO: 33) KSVEESGGRLVTPGTPLTLTCTVSGIDLSTFAMAWVRQAPGKGLEYIGFISLSDATYYATWAKGRFTISKSSSTTVDLKIITPTAEDTATYFCARVVGDSSGYPNTFHPWGPGTLVTVSS

The amino acid sequence of the light chain variable region comprises thefollowing:

(SEQ ID NO: 35) QVLTQTPSPVSAAVGGTVTINCQASQNIHNSDFLAWYQQKPGQPPKLLIYRASKLPSGVPSRFKGSGSGTQFTLTISDLECDDAATYYCQGT YYSGGWYFTFGGGTEVVVK.

The obtained antibodies were tested via Western blot for specificity.Antibody clone D4O5I (Cell Signaling Technologies, Inc.) was used as apositive control. In brief, 10 μg total protein was loaded and separatedon 4-20% SDS-PAGE gel followed by transferring to PVDF membrane. Themembrane was then blocked in TBST with 5% BSA followed by incubationwith 0.2 ug/ml of J89H12L3 and 0.4 ug/ml of D4O5I. Pro epiregulin andits propeptide were detected by goat anti rabbit-HRP conjugate andvisualized by SuperSignal™ West Pico Chemiluminescent Substrate(ThermoFisher Scientific 34079). FIG. 6 demonstrates such a Western blotfor clone J89H12L3. The western blot was negative in negative controlA549 cell line (lane 1) for both clones. Glycosylated (30 kDa) andunglycosylated (17 and 18 kDa) pro-EREG were detected with both clonesin cell lysates from HCC827 (right lane) cells.

The obtained antibodies were also tested in an IHC analysis in xenograftand primary tissue. The IHC was processed with automatic staining system(BenchMark ULTRA, Roche) using the following protocol. FFPE tissuesections were deparaffinized and heated in EDTA antigen retrieval bufferfor 64 min before the rabbit anti-human EREG monoclonal antibodies wereadded to the tissue sections. The incubation time for the rabbit primaryantibody was 16 min at 37° C., and it was followed with a standardOptiView detection protocol from Roche.

FIG. 7 is a comparison between J89H12L3 and D4O5I in various xenografts.Staining is strong in highest EREG expressor − SKE23 cells (A), weak inPLR124EREG +/− cells (B), negative in SK-Hep1 cells (C), and weak inPLR124EREG −/− cells (D) using clone J89H12L3. Similarly clone D4O5Istained SKE23 cells (E) strongly, but it is very weak in PLR124EREG +/−cells (F) and negative for both SK-Hep1 (G) and PLR124EREG −/− cells(H).

FIG. 8 is a comparison between J89H12L3 and D4O5I in lung squamous cellcarcinoma (SCC) tissue. Higher intensity is seen in cancer tissuesstained with clones J89H12L3 (A-C) when compared to those tissuesstained with clone D4O5I (Cell Signaling Technologies, Inc., Danvers,Mass.) (D-F).

FIG. 9 is a comparison between J89H12L3 and D4O5I in lung adenocarcinomaand adenosquamous cell carcinoma. Higher intensity is seen in cancertissues stained with clones J89H12L3 (A-D) when compared to thosetissues stained with clone D4O5I (E-H).

FIG. 10 is an IHC analysis of EREG protein expression in normal andtumor tissues using clone J89H12L3. Moderate to strong staining is seenin skin squamous cell carcinoma (A), hepatocellular carcinoma (B),bladder transitional cell carcinoma (C), colon adenocarcinoma (D), lungadenocarcinoma (E), skin (F), cervix (G), and esophagus (H).

Other Embodiments

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

The invention claimed is:
 1. An antibody or antibody fragment capable ofspecifically binding to amino acids 156-169 of SEQ ID NO: 1, wherein theantibody comprises a heavy chain comprising the following hypervariableregions (HVRs): (a) an HVR-H1 comprising SEQ ID NO: 18; (b) an HVR-H2comprising SEQ ID NO: 19; and (c) an HVR-H3 comprising SEQ ID NO: 20;and wherein the antibody or antibody fragment further comprises a lightchain comprising the following HVRs: (h) an HVR-L1 comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 25 andSEQ ID NO: 26; (i) an HVR-L2 comprising SEQ ID NO: 27; and (j) an HVR-L3comprising SEQ ID NO:
 28. 2. The antibody or antibody fragment of claim1, wherein the heavy chain further comprises the following variabledomain framework regions (FRs): (d) FR-H1 comprising SEQ ID NO: 21; (e)FR-H2 comprising SEQ ID NO: 22; (f) FR-H3 comprising SEQ ID NO: 23; and(g) FR-H4 comprising SEQ ID NO:
 24. 3. The antibody or antibody fragmentof claim 1, wherein the light chain further comprises the followingvariable domain framework regions (FRs): (k) FR-L1 comprising SEQ ID NO:29; (l) FR-L2 comprising SEQ ID NO: 30; (m) FR-L3 comprising SEQ ID NO:31; and (n) FR-L4 comprising SEQ ID NO:
 32. 4. The antibody or antibodyfragment of claim 1, wherein the antibody or antibody fragment comprisesa heavy chain variable region (VH) comprising an amino acid sequencehaving at least 95% sequence identity to SEQ ID NO:
 33. 5. The antibodyor antibody fragment of claim 4, wherein the VH comprises SEQ ID NO: 33.6. The antibody of claim 1, wherein the antibody is a monoclonalantibody.
 7. The antibody of claim 6, wherein the monoclonal antibody isa rabbit monoclonal antibody.
 8. The antibody of claim 6, wherein themonoclonal antibody is an IgG antibody.
 9. The antibody fragment ofclaim 1, wherein the antibody fragment is selected from the groupconsisting of Fab, single chain variable fragment (scFv), Fv, Fab′,Fab′-SH, F(ab′)2, and diabody.
 10. The antibody or antibody fragment ofclaim 1, wherein the antibody or antibody fragment comprises a lightchain variable region (VL) comprising an amino acid sequence having atleast 95% sequence identity to SEQ ID NO: 34 or SEQ ID NO:
 35. 11. Theantibody or antibody fragment of claim 4, wherein the antibody orantibody fragment comprises a light chain variable region (VL)comprising an amino acid sequence having at least 95% sequence identityto SEQ ID NO:
 34. 12. The antibody or antibody fragment of claim 11,wherein the VH comprises SEQ ID NO: 33 and the VL comprises SEQ ID NO:34.
 13. The antibody or antibody fragment of claim 4, wherein theantibody or antibody fragment comprises a light chain variable region(VL) comprising an amino acid sequence having at least 95% sequenceidentity to SEQ ID NO:
 35. 14. The antibody or antibody fragment ofclaim 13, wherein the VH comprises SEQ ID NO: 33 and the VL comprisesSEQ ID NO:
 35. 15. A method of detecting the presence or expressionlevel of human pro-epiregulin in a biological sample comprising:contacting the biological sample with the antibody or antibody fragmentof claim 1; and detecting the presence of the bound antibody or antibodyfragment.
 16. The method of claim 15, wherein the detecting is byimmunohistochemistry, immunofluorescence, or immunoblot.
 17. The methodof claim 15, wherein the biological sample comprises a fixed tissue. 18.The method of claim 17, wherein the fixed tissue is a formalin-fixedparaffin embedded (FFPE) tissue.
 19. The method of claim 15, wherein thebiological sample is from a subject having or predisposed to cancer. 20.The method of claim 19, wherein the cancer is colon cancer, breastcancer, or lung cancer.